3D

BIOPRINTING:
Printing a Brighter Future

• Bioink Selection
• Bioprinting of Tissue Models
• Incorporating Vascularization
• Bioprinting Protocols

Featuring contributions from

Profs. Yu Shrike Zhang, Shaochen Chen,
Yunzhi Peter Yang, and Khoon S. Lim

The Life Science business of Merck operates as

MilliporeSigma in the U.S. and Canada.
Preface
Introduction Current 2D model systems have recognized limitations, such as
3D bioprinting is an increasingly widespread different genotypic and phenotypic cell responses, leading to low
technology with promising applications drug candidate predictability and pre-clinical cell-based assay
in disease modeling, drug discovery, and results. In drug discovery and in-vitro testing, researchers are
regenerative medicine. Successful technology seeking new approaches to overcome some of the limitations
bridges expertise in materials science, of conventional cell culture. Many researchers believe that 3D
engineering, and cell biology, making 3D bioprinting can combine the ease of use of existing cell culture
Megan Muroski, Ph.D methods with the physiological relevance of in vivo animal models
Senior Product bioprinting a well-suited technology for
Manager, Energy and applications in pre-clinical testing and wider and human clinical trials. 3D bioprinting can help to address some
3D Bioprinting of the limitations of 3D cell culture by providing a scalable, and
disease research (Figure 1).
highly reproducible method to form complex 3D structures that
3D Bioprinting enables the creation of functional tissue based on can be automated.
additive manufacturing with improved physiological relevance,
in both pre-clinical and clinical applications. In pre-clinical 3D bioprinting offers the potential of improved tools for disease
applications, 3D bioprinting can be used for in-vitro models research overcoming the current shortcomings in disease models,
and drug discovery, while clinical applications focus on tissue regenerative medicine, and drug discovery. However, in the
regeneration and functional organ replacement. 3D bioprinting development of new 3D bioprinting applications, three main
has the potential to improve the reliability and predictive power of elements must be considered: the bioink, printing method, and
pre-clinical testing through the production of more realistic and application.
reproducible in vitro models.

Extrusion-based bioprinting

Formulate Mix Print

to make an acellular bioink to make a cellular bioink to make a 3D biostructure

Bioprinting

Cross-linker Ready-to-Use
Bioink Cells
Cell-laden
Polymer(s) scaffold
Additives

Other Polymer
ion Polymer (optional)
rat
ife
ol
Pr

Bioink
Cells
and Cells

Human
body
Isolation Cells

Functional peptides
(as growth factors or
adhesion peptides)

Tired of formulations,
try TissueFab™

Figure 1. 3D bioprinting is an additive manufacturing process that uses cells and biomaterials to print an object layer by layer. The steps to create a 3D
bioprinted structure include formulation, mixing, and printing. In formulation, polymers, cross-linkers, and additives are mixed in a matrix to form an
acellular bioink. Cells are simultaneously cultured or isolated and prepared in a solution. Next, the formulated acellular bioink precursor must be mixed
with cells for printing. Common techniques include using a Luer-lock coupler, a static mixer, or dish mixing. Creating the 3D structure by bioprinting is
next. The bioprinter prints the mixture via syringe while following a 3D computer-generated model. After the print is complete, the structure is cured
to prevent dissolution when cell culture media is added. The 3D structure now contains a scaffold and cells, which will grow to fill the 3D printed matrix.
Culture media and additives are required to optimize and enhance cellular expansion.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 1

Table 1. Types of polymers used in bioinks.

Type of Material Examples

Rapid curing, water soluble hydrogel photoinitiators for

visible light polymerization
Initiators & • TPO Nanoparticle
• LAPw
Additives
Traditional photoinitiators
• Irgacure 2959

Mimics bone or stiff tissues

Thermally Low melting point
processable Biodegradable
• Polycaprolactone
polymers
• Polylactide

Includes reactive end groups for hydrogel network

formation and/or functionalization
• Poly(ethylene glycol)
Synthetic • peptides
Polymers • PEG-diacrylate
Reversible gelation
• Pluronic® F-127

Mimics extracellular matrix components

Encourages cell adhesion, growth, proliferation

Natural Examples:
• Sodium Alginate
polymers • Low Endotoxin Gelatin
• Collagen
• Natural Polymer derivatives, e.g. GelMA, HAMA, AlgMA

While the application dictates the choice of cells used, the printing Polymers provide the acellular material in 3D bioprinting that
method should be selected based on the mechanical and physical forms a scaffold and supports cellular growth. Researchers
requirements, such as shear rate, model complexity, and size. have used many different polymers to find the best approach.
Furthermore, the biomaterial ink must have compatibility with the However, polymers alone cannot provide the physical and
cell and properties type to ensure proper printing in the chosen biochemical requirements of cell growth without modification
printer system. In addition, the ink should allow maturation or or formulation with other materials. The most widely used
differentiation of the type of cells used within the construct and biomaterials in 3D Bioprinting include gelatin, alginate, collagen,
be complementary with the downstream analysis. and hyaluronic acid. Alginate, the most popular material used,
derived from brown algae, is biocompatible and provides mild
Bioink crosslinking conditions. Disadvantages of this material include
A bioink is a material that acts as a microenvironment for slow degradation kinetics and poor cell adhesion. Gelatin and
living cells. These materials can contain a variety of polymers, collagen, require chemical modification for crosslinking making
biomaterials, extracellular matrix components, and living cells, high-resolution printing and high fidelity printing challenging,
see Table 1 for the type of polymers used in bioinks. The however, these materials have shown high biological relevance
composition of biomaterial inks can vary in complexity, ranging (bone, skin) and are also popular choices in bioink creation.
from single to multi-component mixtures, and offers varying Hyaluronic acid has biological relevance in connective, epithelial,
levels of modification and crosslinking. Furthermore, biomaterial and neural tissues, but suffers from poor stability.
components can come from various sources, either synthetic or
natural origins, and are further modified to introduce cross-linking
or functionalization sites.
2 Preface

Even with the many tools and customized polymers now available, other biological entities can be sensitive to additive manufacturing
it can still be difficult to make an effective bioink. A good techniques. Both methods offer unique advantages, and the
bioink formulation must balance material properties, printability, printing parameters, biomaterials, and properties of the 3D-printed
biocompatibility, and biochemical cues. On one hand, a bioink constructs will depend on the presence or absence of cells and
material must be sufficiently viscous for printing, but once biological substances.
printed, have the mechanical strength and structural integrity to
maintain its shape. The printed structure must have both high Applications
water content and porosity to enable cells to receive nutrients and 3D bioprinting has the potential to improve the reliability and
oxygen and remove waste and should be soft and biodegradable predictive power of pre-clinical testing through the production of
to allow the cells to spread, migrate, proliferate, and interact with more realistic and reproducible in vitro models. 3D bioprinting can
each other. offer reproducible fabrication of 3D cell-laden constructs to better
mimic conditions in vivo, reducing the need for poorly predictive
In addition to biocompatibility, physical considerations, such
2D models whether in drug discovery, in-vitro disease models,
as viscosity, surface tension, temperature sensitivity must
or regenerative medicine. Vascularization offers significant
also be considered. Currently, there is a need for high-quality,
improvements in our ability to model tissues and disease states
commercially available ready-to-use bioink formulations, such
while at the same time increasing our understanding of critical
as our R&D created TissueFab® bioinks, to enable reproducible
disease pathologies and pathways. Understanding blood vessel
fabrication of synthetic tissues and organs by 3D Bioprinting.
formation and function will develop the field of 3D bioprinting
into pre-clinical testing, broader disease research and precision
Printing
medicine approaches.
There is a wide variety of printing techniques including micro-
extrusion, SLA/DLP, LIFT, inkjet, acoustic, magnetic, and Final Thoughts
volumetric technologies; and of these methods, the most popular
3D Bioprinting is a powerful tool which is enabling the fabrication
being extrusion-based and inkjet printing. Extrusion bioprinting,
of complex tissue and structures for regenerative medicine
in which printing speed and structures can be highly controlled,
and drug discovery. 3D Bioprinting technology is still in its
but shear stress can impact cell viability. Inkjet-based printing is
early stage and several challenges remain to be addressed to
well known for fast printing speed, biological compatibility, and low
move the field forward. Current approaches have had limited
cost, however, requires low viscosity materials.
success for the development of the physical and functional
Each method has specific capabilities making them straightforward components required for tissue regeneration due to their
for a given application while demanding careful selection of inherent complexity in biological, physical, chemical, mechanical
material properties from the bioink used. To this end, optimizing attributes. Newly engineered bioink materials and compatible
the various material and system properties at an early stage will bioprinting applications on the horizon may provide improvements
result in robust and reliable methods for generating tissue and in these challenging areas. Future endeavors in this space will no
disease constructs. doubt increase our ability to model tissues and disease states in
addition to expanding our understanding of disease pathologies
In addition to the type of printing, the technology can be sorted and offering insight into potentially druggable pathways.
into two categories: acellular and cellular constructs. In acellular
bioprinting, the scaffold and biomaterial itself are made in the
absence of cells during the printing process. The advantages
of this technique offer higher accuracy and greater shape
complexity, as the printing criteria can be more rigorous as cell
health does not have to be considered. In cellular bioprinting,
the scaffold and manufacturing are completed in the presence of
cells and other biological agents to mimic living tissue constructs.
Careful consideration must be taken into account as cells and
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 3

Table of Contents
Articles
Design of Bioinks for Bioprinting 5
Xiao Kuang, Guosheng Tang, Zeyu Luo, Yu Shrike Zhang

3D Bioprinting of Functional Tissue Models 20

Min Tang, Shangting You, Jennifer Sun, Wei Zhu,
Shaochen Chen

Hybrid printing for Engineering Vascularized 26

Tissue Constructs
Jiannan Li, Sungwoo Kim, Carolyn Kim, Yunzhi Peter Yang

Recent Advances in 3D Biofabrication of 30

Blood Vessels
Alessia Longoni, Tim BF Woodfield, Jelena Rnjak-Kovacina,
Khoon S Lim

Protocols
Bioprinting Protocol with Ready-to-use 36
TissueFab® Bioinks

Bioink Preparation 38
PhotoCol Methacrylated Collagen Bioink
PhotoHA Methacrylated Hyaluronic Acid Bioink
Lifeink® Type I Collagen Bioink
Lifesupport™ Support Slurry

About this Guide Decellularized ECM Bioink Precursor

INVIVO-Gel
In this guide, we explore 3D bioprinting and its great
potential to improve drug discovery efforts and Cell-bioink Mixing Protocol 51
for implementation at various stages of the drug
General Guide to Photo, Ionic, and Enzymatic 53
development pipeline. We have reviews by leading
Crosslinking
researchers along with protocols designed by leading
R&D experts in Bioprinting.

The guide has been developed for both novice and

Products
experienced researchers interested in 3D Bioprinting. Natural Polymers 56
Our review articles discuss bioprinting and current Cellulose, Chitosans, Ligins, and Hyaluronic Acids
strategies in tissue modeling and vascularization.
As demonstrated in the reviews, biomaterial and
Bioinks 58
TissueFab®, GelMA, Modified Gelatins, Alginate-based Bioinks,
bioprinting options are numerous; however, once a Decellularized Bioinks, Collagen Bioinks, HA Bioinks,
bioprinting application is fully established, it offers Ready-made Bioinks, Biodegradable Polymers, PEG and PEO
excellent reproducibility thanks to the automated
Bioprinting Consumables 61
aspects of the setup. To help get you started, we offer
protocols covering the reconstitution of lyophilized inks,
cell mixing, crosslinking, and bioprinting protocols.
Furthermore, our product guide covers our ready-
to-use, R&D-created, TissueFab® bioinks as well
as popular precursors for advanced users. We hope
that this publication will enable biologists, chemists,
translational researchers, pharma scientists, to explore
the ever-expanding research field of 3D Bioprinting.
We hear you
We now offer a line of low endotoxin,
high-quality ready-to-print bioinks and
bioink precursors.

Cut out your design time with our ready-

to-print bioinks for extrusion-based
bioprinting with products like:

Cat. No. Product Description

918741 TissueFab® - GelMA-Vis-LAP bioink
919926 TissueFab® - crosslinking solution

We also offer bioink precursors to create

your own formulations, such as:
Cat. No. Product Description
919373 Low endotoxin alginate
918628 Low endotoxin GelMA
918644 Low gelatin solution

To find the optimal bioinks and bioink

precursors, please visit
SigmaAldrich.com/bioink-selector

The life science

business of Merck
operates as
MilliporeSigma in
the U.S. and Canada.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 5

Design of Bioinks for Bioprinting

Xiao Kuang, Guosheng Tang, Zeyu Luo, and Yu Shrike Zhang*

Division of Engineering in Medicine, Brigham and Women’s Hospital, Department of Medicine,
Harvard Medical School, Cambridge, MA 02139, United States of America
E-mail: [email protected]
*

Introduction
Bioprinting has emerged as a disruptive biofabrication method for for bioprinting of solid microfibrous and tubular tissues.23–24 Inkjet
producing three-dimensional (3D) tissue constructs.1–2 Bioprinting bioprinting produces droplets from a low-viscosity cell-suspended
potentially enables the customization of fabricated constructs to liquid in a “drop-on-demand” manner to pattern biomaterial inks.
match the unique anatomical structures of a patient, providing Vat (photo)polymerization-based bioprinting refers to bioprinting
personalized 3D representations of tissue structures for applications techniques that use light energy dispersion to manipulate the
in tissue engineering and regenerative medicine.3–5 The typical solidification of photoactive bioinks. Each bioprinting technique
bioprinting process involves the automated deposition of bioinks requires specific physical and chemical bioink properties. The
into target geometries in a layer-by-layer manner, frequently bioink formulation influences the viscosity, surface tension, and
followed by selective material-solidification (i.e., crosslinking). A crosslinking capability, which together determine printability. For
major feature of the 3D bioprinting technology is the use of “bioinks” example, direct extrusion-based bioprinting usually requires high
composed of living cells, extracellular matrix (ECM)-like support viscosity and shear thinning of bioinks to facilitate shape-fixing
biomaterials, and/or other bioactive components to print 3D tissue after material deposition. In situ crosslinking and extrusion in
constructs.6–7 A prerequisite of the bioinks is their compatibility a support bath allow for printing using low-viscosity bioinks.25–26
for the survival of embedded cells. Biomaterial hydrogels with a Inkjet-based bioprinting is limited to low-viscosity bioinks. By
high-water-content environment similar to the physical properties contrast, vat polymerization-based bioprinting requires use of
of the native ECM are extensively used for bioprinting. A variety photocurable bioinks.21–22 More recently, volumetric bioprinting,
of natural polymers, including protein-based materials (e.g., a subgroup of vat polymerization-based bioprinting, enables
collagen, gelatin, fibrin, and silk fibroin), and polysaccharide- layerless, ultra-fast biofabrication.27–29 Besides, other bioprinting
based materials (e.g., alginate, hyaluronic acid (HA)), as well as techniques, such as acoustic bioprinting30 and spheroid fusion-
some biocompatible synthetic polymers (e.g., poly(ethylene glycol) based bioprinting,31 have emerged as parallel innovation tracks of
(PEG)), have been used to formulate boinks.3,8–10 The bioinks both bioprinting techniques and bioink design.
provide different rheological properties, crosslinking behaviors,
The design of customizable bioinks with proper printability
and bioactivities to accommodate successful bioprinting.
(viscosity and crosslinking) and cell viability is central to bioprinting
Up to date, there are several main categories of 3D bioprinting method selection and achieving optimal final tissue construct
techniques, i.e., extrusion-based bioprinting,11–16 inkjet/droplet properties.32–34 This article presents an overview of crosslinking
bioprinting,17–19 and vat polymerization-based bioprinting.20–22 strategies and covers the modification of common biomaterials
Extrusion-based bioprinting, continuously extruding bioinks from for the design of bioinks. The advantages and limitations of widely
a dispensing nozzle, is one of the most versatile bioprinting used biomaterials are compared. In addition, bioink function
techniques. Depending on bioink deposition features, this improvement in terms of printability, bioactivity, and physical
technique can be further divided into direct extrusion, extrusion properties, is summarized. This review provides guidance on the
with bath, and extrusion with microfluidics.13,15 Microfluidics-based bioink design for various bioprinting and biomedical applications.
micro-extrusion using co-axial nozzle systems is extensively used
6 Design of Bioinks for Bioprinting

Overview of Bioink Crosslinking alginate,38–41 gellan gum,42–43 and divalent cations (e.g., Ca2+,
During most 3D bioprinting procedures, aqueous bioinks containing Mg2+, Ba2+) are extensively used in bioprinting due to their fast
living cells and ECM-like support biomaterials are patterned into curing and good biocompatibility. Besides this, electrostatic
target shapes and transformed into solid network structures interaction between oppositely charged polymer chains, such as
via crosslinking. The crosslinking process plays a critical role blends of gelatin-hyaluronate,44 chitosan-alginate,45 and gelatin-
in bioprinting, influencing printing fidelity, mechanical and chitosan,46 also enables hydrogel-gelation. Hydrogen bonding is
physicochemical properties of the bioprinted tissue constructs, a strong intermolecular interaction between hydrogen atoms (in
and cellular behavior after printing.35–37 There are several general amide and hydroxyl) and electronegative atoms (such as oxygen
principles in the design of crosslinking bioinks to consider for and nitrogen). The binding energy of multiple hydrogen bonds
bioprinting: i) biocompatible reagents and end-products for the can be even stronger, which can induce polymer gelation. For
cells, and ii) suitable crosslinking kinetics under physiological example, gelatin and agarose chains can self-assemble upon
conditions (aqueous medium at neutral pH). Depending on the cooling by forming helix complexes via collective hydrogen
nature of crosslinking, both covalent and noncovalent bonding bonds.47–49 Hydrophobic interactions, together with hydrogen
can be used to form a chemical and physical network in bioinks, bonds, contribute to β-sheet folding of silk fibroin for gelation.50–51
respectively. Although some crosslinking processes are not The charge screening of collagen upon pH change can also lead
conducted under physiological conditions, they can still be utilized to hydrogel-gelation.52–53 In addition, host–guest interactions
in bioprinting with optimized reaction conditions to minimize also help to form physically crosslinked dynamic biomaterial
the side effects on the cells. In this section, we summarize the hydrogel.54 Physical crosslinking is usually used to impart shearing
primary crosslinking approaches used in bioprinting (Figure 1). thinning or assist shape-fixing in extrusion-based bioprinting.55–56
It is noted that physical crosslinking can also be sensitive to the
Physical Crosslinking physiological environment and pH levels.
Physical crosslinking takes advantage of weak but collective
Chemical Crosslinking
intermolecular interactions, such as electrostatic interactions,
hydrogen bonding, hydrophobic interaction, and host–guest Chemical crosslinking is extensively used to improve the
interaction, to form a physical network (Table 1). The electrostatic dimension stability and mechanical properties of hydrogels.
interaction between carboxylic acid in polysaccharides, such Chemical crosslinking occurs within functional groups in either

Physical crosslinking Chemical crosslinking Enzymatic crosslinking

Electrostatic interactions Chain growth photopolymerization Glutamine
Metal ion-anions

Tyrosine
Thiol-ene click chemistry
Cationic-anionic charge

Hydrogen bonds
Photo redox

Lysine
Schiff’s base chemistry

Hydrophobic interaction

Acylhydrazone chemistry

CuAAC click chemistry

Host-guest interaction
Glucose
Diels-Alder reaction

Figure 1. Three crosslinking methods and typical examples with involved interactions and reactions for bioink crosslinking. Physical crosslinking relies on
intermolecular interactions, including electrostatic interactions, hydrogen bonds, hydrophobic interactions, and Host–guest interactions. Popular chemical
crosslinking includes chain-growth photopolymerization of methacrylates, and step-growth polymerization using thiol–ene chemistry, photo redox, Schiff’s
base chemistry, acylhydrazone, CuAAC click chemistry, and Diels–Alder reaction. Enzymatic crosslinking of glutamine, tyrosine, lysine amino residues,
and glucose oxidation under relevant enzymes.

250

360
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 7

Table 1. Three major crosslinking methods for common biomaterial hydrogels.

Crosslinking Specific Active Bioink

Notes References
methods mechanism species examples
Fast (+)
Carboxylate/multivalent Alginate, Gellan gum,
Electrostatic interaction pH-sensitive (-) 38,43
cations Agarose

Fast (+)
Gelatin/hyaluronate,
Anionic-cationic moiety Chitosan/alginate, Gelatin/ pH-sensitive (-) 44–46
chitosan
Weak (-)

Gelatin
Physical
Hydrogen bonds Oxygen/hydrogen atoms Tunable binding energy (+) 48–49
crosslinking
Agarose

Weak (-)
Hydrophobic interaction β-sheet-folding Silk fibroin 50–51
Polymer-dependent (-)

Fast (+)
Charge screening Amine Collagen 52–53
pH-sensitive (-)

Additional synthesis (-)

Cyclodextrin/ Cyclodextrin hyaluronate/
Host–guest interaction 54
adamantane adamantane hyaluronate
Weak (-)

Fast (+)
Radical (Meth)acrylates,
Gelatin methacryloyl 58–59
photocrosslinking (meth)acryamides
Free radical (-)

Fast (+)
Thiol–ene chemistry Thiol/vinyl Gelatin–vinyl ester/thiol 62–63
Free radical(-)

Fast (+)
Photoredox Tyrosine, tyramine Silk fibroin 64–67
Chemical Free radical (-)
crosslinking
Fast (+)
Schiff’s base chemistry Aldehyde/amine Gelatin/oxidized dextran 72
pH-sensitive (-)

Fast (+)
Acylhydrazone Oxidized hyaluronate/
Aldehyde/hydrozide 73
chemistry dihydrozide
pH-sensitive (-)

Efficient (+)
CuAAC Click chemistry Azide/alkyne Azide-modified polymers 68–70
Toxic copper (-)

Cell-benign (+)
Transglutaminase Glutamine/lysine Gelatin, Fibrinogen 76–77
Soft (-)

Slow (-)
Horseradish peroxidase Tyrosine, Tyramine Silk fibroin 78–79
Residual H2O2 (-)
Enzymatic
Slow (-)
crosslinking
Lysyl oxidase Glutamine Elastin, Fibroin Noncommercial (-) 81–83

Copper (-)

Heparin-based hydrogels
Glucose oxidase Heparin, Acrylates poly(ethylene glycol)- Slow (-) 84
diacrylate

a step-growth or chain-growth manner, forming a covalent acrylates/(meth)acrylamides and formulated with water-soluble
polymer network. The typical reactions used for chemical photoinitiators to make photocurable bioinks.58–59 A variety
crosslinking of biomaterials are summarized in Table 1. Photo- of water-soluble visible-light triggered photoinitiators, such
triggered chemical crosslinking is the most popular method as lithium-acyl phosphinate (LAP), 2,2’-azobis[2-methyl-N-and
because it is contact-free, and provides spatiotemporal controlled, (2-hydrox-yethyl) propionamide] (VA-086), and eosin Y have been
on-demand, rapid curing.21 For example, the fast chain-growth developed for bioprinting with enhanced cellular viability.21,60 In
photopolymerization reaction of vinyl double bonds is the most the chain-growth photopolymerization, vinyl double bonds on
widely used chemical crosslinking method, particularly in vat the hydrogel-precursors polymerize into carbon-carbon chains
polymerization-based bioprinting techniques.57 Synthetic and by free radicals, forming a heterogeneous network with defects
natural hydrogel precursors can be easily modified with (meth) such as dangling chain, loops, and different chain lengths.61 The
8 Design of Bioinks for Bioprinting

chain-growth crosslinking kinetics are complicated and influenced native ECM, has also been used for enzymatic crosslinking of
by reactive species diffusion and radical termination, such as peptide-based hydrogels. The reaction mechanism reduces lysine
oxygen-inhibition. sidechain residues to form aldehydes, followed by interpeptide
crosslinking with lysine via Schiff’s base reaction.81–83 In addition,
In contrast, the photochemistry of the thiol–ene reaction
glucose oxidase has been used to crosslink PEG-diacrylate
between the thiol and vinyl double bonds proceed in a step-
(PEGDA)-based hydrogels through enzyme-mediated radical
growth manner, forming more uniform networks and showing
polymerization, enabling acrylate-based hydrogel-formation at
less sensitivity to oxygen.62–63 Another increasingly popular
ambient temperature.84
visible light-based crosslinking is the photoredox of tyrosine via
di-tyrosine bond-formation in the presence of ruthenium/sodium Biomaterials as Bioinks
persulfate (Ru/SPS).64 Since many natural proteins contain amino
residues of tyrosine, this crosslinking strategy can proceed in Protein- and Peptide-based Bioinks
the native form of a hydrogel-processor (such as silk fibroin) Collagen is the main structural protein in the ECM of the human
without post-modification.65–66 Meanwhile, hydrogel-precursors body and is rich in connective tissues.83 The most abundant
can be chemically grafted with the tyramine groups to facilitate collagen protein is collagen type I. Collagen possesses tissue-
di-tyrosine-formation.67 Besides this, various other biorthogonal matching physicochemical properties and excellent in vitro/in vivo
click chemistry, including azide-alkyne cycloadditions,68–70 biocompatibility, and is widely used in biomedical applications.85–86
Diels–Alder [4+2] cycloaddition,71 Schiff’s base chemistry,72 and Collagen is soluble at low temperatures (2–8 °C) and acidic
acylhydrazone chemistry73 can also be exploited for biopolymer conditions and forms a fibrous gel by changing to neutral pH.
crosslinking.74 However, most of these crosslinking approaches Collagen can also self-assemble at 37 °C, enabling slow gelation.6
involve the post-modification of the hydrogel-precursors. Due to these reasons, pure collagen is not directly used as
bioinks. Instead, it is most often used as the cell support layer
Enzymatic Crosslinking in bath–based bioprinting.53 Properties of collagen bioinks can
Besides normal curing reactions using additional chemicals be tuned by simply blending with other biomaterials, such as
or crosslinkers, enzymes are also utilized to catalyze the alginate,87 agarose,88 and silk,89 to enhance the printability. In
crosslinking of protein-based bioinks at physiological conditions.75 addition, collagen can be chemically modified with photosensitive
Transglutaminases can catalyze the formation of isopeptide bonds groups, such as methacryloyl, for light-based crosslinking.90–91
between lysine ε-amines and glutamine sidechain amides.76–77 For example, a multicomponent bioink containing methacryloyl-
This reaction has been used to effectively crosslink proteins modified collagen and thiolated hyaluronic acid was developed
bearing rich lysine and glutamine residues, such as fibrinogen to print tissue models to mimic the liver microenvironment.90 In
and gelatin, to produce insoluble hydrogels.75 Another widely used another study, methacryloyl-modified collagen was blended with
enzyme system is the hydrogen peroxidase (HRP) for hydrogel alginate to bioprint a human corneal model.91 With the combined
crosslinking via tyramine oxidative coupling in the presence of control over pH, temperature, collagen ratios, and precursor
hydrogen peroxide (H2O2). Tyramine-rich hydrogel-precursors, concentration, the gelation and printing fidelity of collagen and
including natural silk fibroin78–79 and tyramine-functionalized HA,80 derivatives can be tuned to facilitate successful printing.92–94
can be effectively crosslinked by this HRP/H2O2 system. Lysyl Collagen and typical derivative-based bioinks for bioprinting are
oxidase, a critical enzyme for the formation and repair of the summarized in Table 2.

Table 2. Summary of natural and synthetic polymers and their derivatives for bioinks using different crosslinking methods.

Primary Printing Crosslinking

Bioink Notes References
material approach method

Col Extrusion Neutralize pH change 53

Improved printability and cell-

Col/Fib Extrusion Neutralize before print 229
adherence
Collagen
Col-MA/Alg Extrusion Ionic crosslinking Increased mechanic property 91

Col-MA/HA Extrusion UV irradiation after bioprinting Light curable 90

Ionic crosslinking +
Gel/Alg Extrusion Increased mechanical properties 230
glutaraldehyde

Extrusion
Improved printability and cell-
GelMA Photocrosslinking 12,101
adherence
Vat-polymerization

Improved printability and cell-

D-GelMA Vat-polymerization Photocrosslinking 231
adherence
Gelatin
Ionic crosslinking +
GelMA/Alg Extrusion Enhanced printability 199,232
photocrosslinking

Gel-NB Vat-polymerization Photocrosslinking Low swelling ratios 233

GelMA-Tyr Extrusion Photocrosslinking Enhanced adhesion 67,234

Extrusion
Gel-AGE Thiol–ene + photoredox Low bioink viscosity 62,235
Vat-polymerization
 3D Bioprinting: Printing a Bright Future
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Primary Printing Crosslinking

Bioink Notes References
material approach method

Slow curing
Extrusion HRP/H2O2 128
SF residual H2O2
Silk fibroin
Extrusion Ru/SPS Fast crosslinking 66

SF-MA Vat-polymerization Photocrosslinking Fast crosslinking 127,236

dECM Extrusion Physical crosslinking at 37 oC Mechanically weak 131

dECM dECM-MA Extrusion Photocrosslinking Mimicking native microenvironment 90

dECM/Alg Extrusion Ionic crosslinking Good printability 237–239

PEG monoacrylate/
Ionic crosslinking + photo- Increased mechanical property and
Fibrinogen Extrusion 134
irradiation during bioprinting printability
Fib/Alg

MeTro/GelMA Extrusion Photocrosslinking High elasticity 138

Elastin
MeTro/CNT Extrusion Photocrosslinking Enhanced conductivity 137

Extrusion
Alg Ionic crosslinking Fast curing, low cell adherence 240–241
Inkjet

Ionic crosslinking
Alg-MA Vat-polymerization Dual-crosslink 242–243
Photocrosslinking

Extrusion Ionic crosslinking

Alginate Oxi-Alg-MA Rapidly degradable 244–245
Vat-polymerization photocrosslinking

Ionic crosslinking
Alg-NB Extrusion Tunable properties 147
photocrosslinking

Alg-Tyr Extrusion HRP/H2O2 Fast curing 149

Alg-RGD Extrusion Ionic crosslinking Improve cell adhesion 153,246

HA Extrusion - Weak 247

Extrusion
HA-MA Photocrosslinking Enhanced stability 164–166
Vat-polymerization

Extended degradation time, increased

Oxi-HA Extrusion Schiff’s base 170,173–174
Hyaluronic stability
acid
Short term stable,
HA-ADH/Oxi-HA Extrusion Acylhydrazone chemistry 171–172
self-healing

HA-Tyr Extrusion HRP/H2O2 High cell viability 80,149

HA-CD/HA-Ad Extrusion Host–guest interaction Self-healing 54

PEGDA Vat-polymerization Photocrosslinking Tunable properties 182–183

PEGX Extrusion Photocrosslinking Low viscosity, tunable curing chemistry 184–185

PEG
Ionic crosslinking +
PEGDA/Alg Extrusion High toughness 228
Photocrosslinking

F127 Extrusion Thermal gelation Temperature sensitive 189–192

Pluronics Extrusion
F127DA Photocrosslinking Low cell adherence, high toughness 248–249
Vat-polymerization

Abbreviations: collagen (Col), methacryloyl-modified collagen (Col-MA), gelatin (gel), gelatin methacryloyl (GelMA), dopamine-modified gelatin methacryloyl
(D-GelMA), allylated gelatin (Gel-AGE), vinyl ester modified gelatin (Gel-VE), norbornene-modified gelatin (Gel-NB), cysteine-modified gelatin (Gel-Cys), tyramide-
modified gelatin (Gel-Tyr), furan-modified Gelatin (Gel-FA), furfuryl-modified gelatin (Gel-FI), silk fibroin (SF), methacrylated silk fibroin (SF-MA), decellularized
extracellular matrix (dECM), methacryated decellularized extracellular matrix (dECM-MA), fibrinogen (Fib), methacryloyl-modified tropoelastin (MeTro), alginate (Alg),
methacrylated alginate (Alg-MA), norbornene-modified alginate (Alg-NB), tyramide-modified alginate (Alg-Tyr), RGD-modified alginate (Alg-RGD), oxidized alginate
(Oxi-Alg), hyaluronic acid (HA), methacrylated hyaluronic acid (HA-HA), oxidized hyaluronic acid (Oxi-HA), tyramide-modified hyaluronic acid (HA-Tyr), norbornene-
modified hyaluronic acid (HA-NB), azide-modified hyaluronic acid (HA-Azide), dihydrazide-modified hyaluronic acid (HA-ADH), furan-modified hyaluronic acid (HA-
FA), β-cyclodextrin-modified hyaluronic acid (HA-CD), adamantine-modified hyaluronic acid (HA-Ad), polyethylene glycol-diacrylate (PEGDA), Pluronic F127 (F127),
Pluronic F127-diacrylate (F127DA), ruthenium/sodium persulfate (Ru/SPS).
10 Design of Bioinks for Bioprinting

A) Gelatin(Gel) Pro Lys(4%) Gly Glu(10%) Hyp(12%) Gly Pro B) Coli

Heat Cool

Triple helix

C) (Meth)acrylamide/(meth)acrylate Ene derivatives Thiol derivatives Tyramine derivative Furan derivatives

Gel-MA Gel-AGE Gel-Cys Gel-Tyr Gel-FA

Figure 2. Gelatin and derivatives with different crosslinking for bioinks. A) Chemical structures with major amino residues and typical reactive functional
groups in gelatin. B) Gelatin chains reversibly transform from random coil to triple helix configuration upon heating/cooling modulated by hydrogen bonds.
C) Major gelation derivatives: methacrylated gelatin (Gel-MA), ene-derivatives including allylated gelatin (Gel-AGE), vinyl ester-modified gelatin (Gel-VE),
norbornene-modified gelatin (Gel-NB), thiol-modified gelatin including cysteine-modified gelatin (Gel-Cys) , and thiobutyrolacton-modified gelatin (Gel-
Thiol), tyramide-modified gelatin (Gel-Try), furan/furfuryl-modified gelatin (Gel-FA, Gel-FI). 250

Gelatin is produced by hydrolyzing collagen from connective have been grafted on gelatin molecules.104–105 360For example,
tissues of animals, such as the skin.95 The primary reactive pentenoate-modified gelatin,104 vinyl ester-modified gelatin (Gel-
functional groups of gelatin are amine, carboxylate, and hydroxyl VE),106 norbornene-modified gelatin (Gel-NB),63,107 and allylated
groups (Figure 2A). Similar to collagen, gelatin contains cell- gelatin (Gel-AGE)62 can be modified and then photocrosslinked
adhesive peptide sequences, such as Arg-Gly-Asp (RGD) as with multifunctional thiol crosslinkers. Thiol functionalized 450
gelatin,
well as protease-sensitive sites. It has been approved by the including cysteine-modified gelatin (Gel-Cys),104,108 and gelatin
United States Food and Drug Administration (FDA) for biological thiobutyrolacton,104 were also used for thiol–ene chemistry or
and biomedical applications.96 Due to the advantages of good thiol-Michael reactions. Furan-modified gelatin (Gel-FA) was
bioactivity, biocompatibility, biodegradability, and low cost, as synthesized for Diels-Alder reaction-based crosslinking.109 Besides,
well as widely tunable properties, gelatin is one of the widely Gel-FA can proceed with photo-oxidation-based crosslinking in
used materials in tissue engineering, and bioprinting.61,97 There the presence of a photosensitizer, such as Rose Bengal.110–111
are several methods to crosslink gelatin-based biomaterials. Moreover, the wide use of gelatin and its derivatives to blend with
As shown in Figure 2B, gelatin displays a thermally reversible other biomaterials, such as alginate and PEGDA, increases the
sol-gel transition upon heating and cooling due to its reversible printability and physical properties of the bioinks.112–113
triple helix-coil transition.98–99 To improve the structural integrity
Silk fibroin is a natural protein derived from silkworms
of gelatin hydrogels for in vivo applications, chemical crosslinking
(Figure 3A),114 which has attracted increasing attention for
of gelatin by glutaraldehyde, genipin, and 1-ethyl-3-(3-dimethyl
use in bioprinting.115–117 The most widely used silk material is
aminopropyl) carbodiimide (EDC) are used.100 In addition, more
the domesticated Bombyx mori silk protein, which shows good
benign enzymatic crosslinking of gelatin using transglutaminase
biocompatibility, low inflammatory profile, and tunable mechanical
is extensively adopted.75–76 Meanwhile, various gelatin derivatives
properties.118 Silk fibroin consists of hydrophobic segments with
have been developed for fast chemical crosslinking (Figure
multiple hydrogen bonds and hydrophilic segments containing
2C).96 For example, the formation of (meth)acrylamide- and
reactive amino residues of tyrosine (5.2%), lysine (0.3%), and
(meth)acrylate-modified gelatin (GelMA, or gelatin methacryloyl)
glutamate (1.0%).119–120 To prepare silk fibroin–based bioink, silk
through reaction between amine (in lysine) and hydroxyl (in
fibroin is treated with ionic liquids or water-based salt systems
hydroxyproline) residues with methacrylate anhydride (MAAH)
to break the strong hydrogen bonds between the proteins.121 The
to form pending vinyl double bonds is the most widely used
silk fibroin hydrogels can be obtained using chemical and physical
derivatives for chain-growth photopolymerization.12,58,97,101–103 Also,
crosslinking approaches. Physical crosslinking of silk fibroin is
the carboxyl acid residues (glutamate) can be modified with
achieved through a structural transformation from a random-coil
tyramine for photo-reduction by the Ru/SPS system.67 In addition,
configuration to β-sheet folding (Figure 3B),122–123 which can be
step-growth polymerization by click chemistry, including thiol–
accelerated by treatment conditions, including sonication, shear
ene chemistry, Diels–Alder reaction, and Schiff’s base, is widely
action, temperature change, pH adjusting, electrical stimulation,
used for crosslinking gelatin hydrogels.61 To develop thiol–ene
and addition of polar organic solvents, such as polyols or
photo-crosslinkable gelatin, ‘ene’ functional groups, including
surfactants.124 The physically crosslinked silk hydrogels with a
norbornene, vinyl esters, pentenyl, allyl ethers, or acrylates,
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 11

A) Silk fibroin (SF) A) Alginate(Alg) B)

β-D Mannuronic acid (M)

a-L guluronic acid (G)

C) Alg-MA Alg-NB Oxi-Alg

B) Silk β-sheet folding C) Silk di-tyrosine Methacrylate derivatives

Silk-MA

HA-Tyr Alg-RGD

Figure 3. Silk fibroin and derivatives with different crosslinking for bioinks.
A) Chemical structures with major amino residues and typical reactive
functional groups in silk fibroin. B) Physical crosslinking of silk fibroin Figure 4. Alginate and derivatives with different crosslinking for bioinks.
by configuration transformation from random coil to β-sheeting folding A) Chemical structure of alginate. B) Electrostatic interactions between
dominated by the collective hydrogen bonds in the hydrophobic segments. metal ions and carboxylate for ionic crosslinking of alginate. C) Typical
C) Chemical crosslinking of SF: native silk fibroin via di-tyrosine-formation 250
alginate derivatives: methacrylated alginate (Alg-MA), norbornene-
and methacrylated silk fibroin (SF-MA) for photocrosslinking. modified alginate (Alg-NB), tyramide-modified alginate (Alg-Tyr), RGD-
modified alginate (Alg-RGD), oxidized alginate (Oxi-Alg). 250
large amount of β-sheet folding are usually stiff and hard to be
360
remodeled by cells. To this end, soft silk fibroin hydrogels are Polysaccharide-based Bioinks
fabricated by chemical crosslinking. The native silk fibroin can be Polysaccharides and their derivatives have attracted considerable
attention as biomaterials for use in biomedical engineering and
crosslinked by forming di-tyrosine bonds between the phenolic 450
tyrosine residues (Figure 3C). The di-tyrosine–based crosslinking materials science. Here, we briefly introduce polysaccharides,
is realized by either photoredox-based chemical crosslinking, including alginate, HA, agarose, chitosan, and their derivatives as
64,125
or enzymatic crosslinking approaches using HRP/H2O2.79,126 bioinks. Alginate, also known as alginic acid, is a natural anionic
Particularly, the photoredox crosslinking using Ru/SPS system polysaccharide refined from brown seaweed composed of β-d-
triggered by visible light is efficient and rapid,64 and can be used mannuronic (M) and α-l-guluronic acids (G) (Figure 4A), which
in bioprinting of silk fibroin.66 In addition, silk fibroin can also be is similar to the glycosaminoglycans found in the native ECM of
modified with methacrylate groups for vat photopolymerization- the human body. It has seen significant use in biomedicine and
based printing (Figure 3C).127–128 tissue engineering due to its biocompatibility, low cytotoxicity,
mild gelation process, and low cost.139–140 In particular, it has
Other proteins, including decellularized ECM (dECM), fibrin, and been widely used as a bioink because of its rapid gelation
elastin, are also used for bioinks. The dECM is obtained from without harmful byproducts.3 As shown in Figure 4B, ionic
the target tissue with all the cellular components removed while gelation of alginate can be easily achieved by rapid crosslinking
preserving the proteins, and gross architecture can also be of the carboxylic acid group in the G unit by divalent cations,
preserved as needed.129 The dECM hydrogels are rich in ECM such as calcium (Ca2+), to form an “egg-box” structure.141
components, improving cell viability and functionality by providing Thus, bioinks based on alginate, modified alginate, or alginate
a natural microenvironment. The dECM pregel solutions are liquid blended with other biomaterials are some of the most widely
below 10 °C and slowly gelates above 37 °C.130 Despite these used in bioprinting.40–41 Unfortunately, ionic crosslinking of
advantages, the low mechanical strength of dECM limits its wide alginate is not stable under physiological conditions due to ionic
application. For this reason, dECM is frequently modified by exchange.142 Accordingly, various alginate derivatives, such as
incorporating photosensitive functional groups or mixed with other methacrylated alginate (Alg-MA), are synthesized for chemical
cross-linkable components when used in bioinks.131 Fibrin exhibits crosslinking (Figure 4C). Alg-MA was obtained by reaction
good biodegradability and can promote cell growth and tissue with MAAH,143 glycidyl methacrylate (GMA),144 or 2-aminoethyl
regeneration.132 However, the high viscosity of fibrin makes it methacrylate in the presence of EDC and N-hydroxysuccinimide
difficult to use without modification as a bioink in extrusion-based (NHS).145–146 Rapid UV-induced thiol–ene crosslinking is used to
3D bioprinting.133 As a result, fibrin is typically chemically modified crosslink norbornene-modified alginate (Alg-NB).147–148 Additionally,
and/or blended with other printable materials such as gelatin to enzymatic crosslinking of tyramide-modified alginate (Alg-Tyr)
make bioinks.134,135 Elastin can provide elasticity when used as a by HRP/H2O2 is utilized for extrusion bioprinting.149 Derivatives
major component for printing of tissues like skin,136 but its intrinsic of alginate with in-vivo biodegradability are also highly sought
crosslinking creates challenges to using it as a bioink. Therefore, after for biomedical applications.150 To enhance biodegradability,
its soluble precursor, such as recombinant tropoelastin, has been oxidized alginate (Oxi-Alg) was prepared by partially breaking
used to produce bioinks.137–138 the ring using a strong oxidant, such as sodium periodate, and
tuning the degradation rate by the oxidation degree.145,151 Alginate
12 Design of Bioinks for Bioprinting

also lacks cell adhesion sites, leading to poor cell proliferation based hydrogels by host–guest interaction,54 oxidized-HA and
and differentiation. To this end, cell adhesive peptides of RGD dihydrazide-modified HA (HA-ADH) dynamic hydrogel by Schiff’s-
can be conjugated to alginates.152–153 Thus, alginate bioinks can base chemistry,170–174 and dynamic thiol-HA/silver composite
be engineered with a wide variety of functionalities, including hydrogel,175 to assist the material extrusion or enable functional
photocrosslinkability, biodegradability, and cellular attachment self-healing properties. HA can blend with gelatin components
properties for various biomedical applications.154–155 Further, multi- or be modified with RGD to improve cellular interaction.165,174
component hydrogels can be created by blending alginate with Therefore, these HA modifications enable the fabrication of
other polymers, including chitosan, gelatin, and hyaluronic acid to mechanically stable, biodegradable hydrogels with good cell
enhance comprehensive performance.156–158 adhesion for bioprinting. Overall, derived HA with modified
properties has been widely used as bioinks in tissue engineering,
HA is a non-sulfated glycosaminoglycan that is ubiquitous in the
regeneration medicine, and biomedical applications.176–178
human body, especially in skeletal structures and supporting
tissues, like skin, bone, cartilage, and vascular tissue.59,159 Besides these, other polysaccharides and derivatives are used
HA mediates cellular signalling, and is a critical component as bioinks as well. Agarose is a biocompatible polysaccharide
of synovial fluid, vitreous humour, and hyaline cartilage.160 extracted from marine algae and seaweed. Agarose shows
Due to its excellent biocompatibility, HA hydrogels have been thermoreversible gelation at around 30–40 °C depending on the
progressively applied in biomedical applications for decades.161–162 aqueous solution concentration and molecular weight, and is
However, when used in bioinks HA provides limited capability for suitable for extrusion-based printing of structures.179 Similar to
cell migration, angiogenesis, and proliferation, as well as weak other polysaccharides with poor cell adhesion, agarose can be
mechanical properties.163 Although high molecular-weight HA mixed with additional components, such as gelatin or collagen, to
is viscous and displays shear-thinning properties, it is difficult prepare bioinks.180 Besides, agarose can be chemically modified
to maintain its printed shape and is mechanically weak in an by carboxylation to soften the hydrogel by reducing the helical-
uncrosslinked HA hydrogel. To successfully use HA as a bioink, helical interactions and/or graft RGD to enhance cell adhesion.180
various chemical modifications must be employed to incorporate Chitin is the most naturally abundant amino-polysaccharide
reactive functional groups for crosslinking (Figure 5). This is with biocompatible and biodegradable properties.181 By partial
accomplished via reaction of the three major functional groups: deacetylation of chitin, chitosan is obtained with amine residues
glucuronic acid, and the primary and secondary hydroxyl groups. that show good anti-bacterial and wound healing performance.181
For example, methacrylated HA (HA-MA) can be obtained by Chitosan can be readily dissolved in dilute acids for facile
reacting HA with MAAH or GMA for light-trigged hydrogels processing, and further blended with other polymers to prepare
crosslinking.164–166 In addition, several other HA derivatives have multi-component bioinks.45–46
been developed to prepare HA hydrogels by different crosslinking
approaches, including thiol modified HA (HA-Thiol) for thiol- Synthetic Polymer-based Bioinks
Michael addition,167–168 norbornene-modified HA (HA-NB) for Synthetic polymer hydrogels formed by chemical crosslinking of
thiol–ene reactions,169 and tyramide-modified HA (HA-Try) for synthetic monomers or oligomers have also been widely used
enzymatic crosslinking,80,149 furan-modified HA (HA-FA) for Diels– for bioprinting.8 Compared with natural polymer hydrogels,
Alder crosslinking,71 and azide-modified HA (HA-Azide) for CuAAc synthetic polymer hydrogels have stronger and more highly
click chemistry.70 Meanwhile, HA was also modified to fabricate tailorable mechanical properties. Among them, the most widely
hydrogels containing dynamic bonds, including β-cyclodextrin- used synthetic hydrogel is based on PEG or poly(ethylene
modified HA (HA-CD) and adamantane-modified HA (HA-Ad) oxide) (PEO) (high molecular weights, >10 kDa) hydrogels.

Hyaluronic acid (HA) HA-MA HA-Thiol HA-Tyr

Glucuronic acid
N-acetyl Glucosamine HA-NB HA-Azide

Oxi-HA

HA-FA HA-CD HA-Ad

HA-ADH

Figure 5. Chemical of hyaluronic acid and typical derivatives: methacylated HA (HA-MA), HA-Thiol, oxidized HA (Oxi-HA), norbornene-modified HA
(HA-NB), tyramide-modified HA (HA-Tyr), azide-modified HA (HA-Azide), dihydrazide-modified HA (HA-ADH), furan-modified HA (HA-FA), β-cyclodextrin-
modified HA (HA-CD), and adamantane-modified HA (HA-Ad).

250
 3D Bioprinting: Printing a Bright Future
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A)

PEGDA
PEG
PEG 4-Arm-X

B)

PEO-PPO-PEO(Pluronics F127) F127DA

T>LCST

T<LCST

Figure 6. Chemical structure, modification of synthetic polymers to prepare crosslinked bioinks A) PEG and derivatives. B) Pluronic F127 and derivatives.

250
PEG contains hydrophilic ethylene oxide repeating units and proteins, and a lack of biodegradability. To this end, various active
hydroxyl end-groups. PEG can be tailored to have different components, such as cell-adhesive peptides or growth factors,
360
can be incorporated into synthetic hydrogels, as discussed later.
molecular weights and chain architectures, including linear or
multi-arms. To accommodate crosslinking for bioprinting, PEG
can be modified with (methyl)acrylate, such as PEGDA.182–183 Bioink Functional Improvements
Under light irradiation, PEGDA can proceed with chain-growth Printability 450
photopolymerization with the aid of various photoinitiators
Printability of bioinks generally refers to the degree of extrudability,
(Figure 6A). By altering the oligomer concentration and oligomer
filament uniformity, and shape fidelity of the printed object
chain length, the mechanical properties of PEGDA hydrogels can
compared to the original computer-aided design.194 Printability
be simply tuned in a wide range. PEG has also been modified
is closely associated with multiple rheological properties (e.g.,
with other functional groups, including carboxylic acid, vinyl,
bioink viscosity and viscoelasticity), physical properties (e.g.,
amine, and thiol groups for step-growth polymerization with click
surface tension), crosslinking strategies as well as bioprinting
chemistry-based crosslinking.184–185 It is noted that the existence
parameters (e.g., applied pressure). Usually, bioinks with a high
of low-molecular-weight PEGDA residual oligomers would lead to
viscosity and shear-thinning enable good printing fidelity in direct
cytotoxicity in PEGDA hydrogels.186
extrusion-based bioprinting. Various biocompatible rheological
Another class of extensively studied synthetic polymer is modifiers, such as agarose,179 gelatin,195, and laponite,196–197 are
Poloxamer (or Pluronic F127), which is a linear triblock copolymer widely used to tune the rheological properties of bioinks. However,
composed of hydrophilic PEO and hydrophobic poly(propylene a bioink with high viscosity and a large shearing force in the
oxide) blocks (PEO−PPO−PEO). Pluronic F127 has been approved dispensing nozzle can also be detrimental to living cells.198 In this
by FDA as a human-use cytocompatible biomaterial.187 It sense, the use of low-viscosity bioinks with in situ crosslinking
shows polymer concentration-dependent thermoreversible sol− and support bath-based extrusion are favored in specific
gel transition. Above a critical micelle concentration and a scenarios.25–26 Another way to enhance the printability of a bioink
temperature in the range of 4−10 °C, Pluronic F127 reversibly is to include a secondary component that uses rapid physical
forms a physical gel via micellar aggregation (Figure 6B). The crosslinking as a sacrifice template.142 Low-viscosity alginate is
concentration of Pluronics F127 must be as high as 14 % to form extensively mixed with GelMA,199–200 collagen,142 and dECM113 and
a gel in a cell culture medium, leading to low cell viability.188 used as a sacrifice template for co-axial extrusion bioprinting.
Therefore, Pluronics F127 is more frequently used as a sacrificial For example, we reported a general method for bioink design
or support material in extrusion-based bioprinting.189–192 Acrylated using alginate-templated dual-stage crosslinking for use with
Pluronic F127 (F127DA) was synthesized to produce photocurable low-viscosity bioinks.142 These multi-component bioinks consisted
bioinks.188,193 Significant drawbacks of synthetic hydrogels include of low viscosity alginate combined with bio-macromolecular
their biological inertness, inherently poor adhesion for cells or components such as collagen, gelatin, or GelMA. The fast ionic
crosslinking of the alginate component serves as a temporal
14 Design of Bioinks for Bioprinting

A) B) (i) Day 0 (ii) hMSC

Composite bioink
-Alginate
-Biomacromolecule

CaCI2
Bioink
Templated network 50 µm
-Physical crosslinking of
alginated by Ca2+
(iii) Day 1 (iv) Day 5

Bioprinting

Biomacromolecule network
-Chemical/physical crosslinking
of biomacromolecule
-Removal of alginate through
monovalent ion exchange or
Wash & Culture using Ca2+-chelator 10 µm

C) (i) Bioink (ii) 3D Bioprinting (iii) Cell Spreading D) (i) (ii)

50 µm 50 µm

(iii) (iv)

Pre-gel Crosslinked Non-spreading Spreading

solution GeIMA PEO cell cell
100 µm 100 µm

Figure 7. Typical examples of bioink-customization to improve printability, cell viability, and mechanical properties of the bioprinted constructs. A)
Schematic showing alginate-templated dual-stage crosslinking for bioprinting. The multi-component bioink contains low-viscosity alginate with fast ionic
250
crosslinking and biomacromolecule components with secondary crosslinking for co-axial bioprinting. B) Photographs of bioprinted five-layer constructs
at day 0 and day 1 post-bioprinting, as well as fluorescence microscopic images of live/dead staining (live cells in green and dead in red) at day 0, and
F-actin/nuclei staining (F-actin in green and nuclei in blue) at day 5 of the encapsulated hMSCs in the bioprinted constructs. A) and B) are adapted with
permission from reference 142, copyright 2018 John Wiley and Sons. C) Schematic showing 3D bioprinting of a micropore-forming hydrogel structure using
the APTE bioink and a conventional hydrogel structure. D) Confocal fluorescence micrographs showing morphologies of NIH/3T3 fibroblasts within i, iii)
standard GelMA constructs and ii, iv) microporous GelMA constructs. The cells were stained for nuclei (blue) and F-actin (green). C) and D) are adapted
360
with permission from reference 207, copyright 2018 John Wiley and Sons.

structural support to stabilize the construct shape during co-axial 450

this issue. In the case of vat polymerization-based bioprinting,
extrusion bioprinting. After chemical or physical crosslinking of exposure to light irradiation can also impact the living cells. Thus,
the bio-macromolecular component, the alginate physical network highly efficient visible-light photoinitiators are favored to initiate
was selectively removed to leave the desired bio-macromolecule crosslinking.21 After printing, cell growth can also be influenced
network (Figure 7A), enabling good cell viability and spreading in by hydrogel bioactivity. Therefore, hydrogels that possess cell-
the resulant hydrogels (Figure 7B). attachment sites, such as gelatin and dECM, are frequently
used alone or mixed with other components. To enhance the cell
Enhancing Cellular Behaviors adhesion and bioactivity of synthetic polymer-based hydrogels,
The cellular behavior of bioprinted tissue constructs can be biologically active components, such as cell-adhesive RGD
influenced by many factors before, during, and after printing.201 peptides or growth factor-sequestering heparan sulfate proteins
First, before printing, cell-laden bioinks containing hydrogel- (HSP) can be grafted onto polymer chains to prepare the bioinks.
precursors, initiators, or crosslinkers must be biocompatible For example, Moon et al. developed PEG hydrogels with integrin-
with cells. Hydrogel systems that use toxic chemical crosslinkers binding sites and protease-sensitive substrates to produce bioinks
or extreme pH conditions are not suitable candidates for use with good cell adhesion and biodegradability.203
in cell-laden bioinks. For extrusion-based bioprinting, overly
Besides the cell adhesion sites, the polymer network structure
high shearing stress can cause cellular deformation, leading to
can also influence cell growth. Biomaterial hydrogels with
cell damage or loss of function.202 Use of lower shearing force,
high polymer concentration or crosslinking density featuring
lower-viscosity bioinks, or shear-thinning bioinks can mitigate
small network mesh sizes reduce cell viability and retard cell
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spreading and proliferation.204–205 However, increasing the mesh with tuned mechanical properties.216–219 In addition, multi-walled
size to enhance cell viability sacrifices mechanical properties. carbon nanotubes (MWCNTs) have also been added to GelMA to
To address this, micro pore-forming hydrogel biomaterials have enhance both the mechanical and electrophysiological properties.
been developed that can be applied to bioprinting.206–208 For Magnetic nanoparticles have also been added to hydrogel matrices
example, a micro pore-forming bioink based on the aqueous to introduce magnetic properties.220–222 Interpenetrating polymer
two-phase emulsion (ATPE) improved cell viability and enhanced network hydrogels, specifically double-network (DN) hydrogels,
cell proliferation (Figure 7C).207 The ATPE bioink was prepared have attracted attention for increasing the mechanical properties
by mixing GelMA and PEO aqueous solutions, forming phase- of the hydrogels, especially the toughness.223–224 DN hydrogels
separated PEO domains in the range of several tens of consisted of one stiff and brittle polymer network and a second
micrometers. The cell-laden ATPE bioink was used for extrusion- soft and ductile network. This concept is also gaining popularity
based bioprinting or vat polymerization-based bioprinting for fabricating tough biomaterial hydrogels. To design bioinks
modalities. After photocrosslinking of the GelMA component, for tough biomaterial hydrogels, an ionically cross-linkable
the PEO microphase was leached, forming microscale pores. The polysaccharide, such as alginate,225–227 is suitable for the stiff first
3D-bioprinted porous GelMA constructs, encapsulating various network; loose chemical crosslinking is adopted for the second
cell types, showed better viability, proliferation, and metabolic biopolymer network. For example, Hong et al. developed a DN
activity than those bioprinted with conventional pure GelMA as hydrogel-based bioink containing high-molecular-weight PEGDA
the bioink (Figure 7D).101,209 Bioprinted 3D tissues with enhanced and medium-viscosity sodium alginate.228 To facilitate extrusion
cellular behaviors such as these may soon become widespread in bioprinting, nanoclays were also added to impart shearing. After
tissue engineering and tissue model engineering.210–212 crosslinking, the hydrogel with a water content of 77.5 wt% had
a fracture toughness above 1,500 J m-2. The ionically crosslinked
Physical Properties alginate dissipated mechanical energy under deformation,
Several physical properties must be considered for the clinical and the long-chain PEG network maintained high hydrogel
translation of bioprinted tissue constructs including biodegradability elasticity. Since both PEG and alginate are biocompatible, this
and mechanical properties. Human tissue has unique mechanical tough hydrogel enabled high viability of encapsulated human
properties, including low modulus, viscoelasticity, high toughness, mesenchymal stem cells (hMSCs) for up to 7 days.
and good fatigue-resistance.213 Bioprinted tissue constructs require
comparable stiffness to match that of the targeted tissue and Conclusions
must show controllable degradation to remodel with the host Bioprinting is a disruptive biofabrication technique that allows
tissue. It is highly demanding to have both widely tunable and for direct fabrication of anatomical 3D tissue constructs based
good mechanical properties in bioprinted constructs. Therefore, on individual patient needs. With the advances in bioprinting
the design and optimization of bioprinting parameters must be techniques and bioink design, the bioprinting of complex cell-laden
carefully customized. Take GelMA as an example; mechanical tissue constructs has found broad applications in tissue modeling
properties of a GelMA hydrogel can be altered by changing GelMA and regenerative medicine. Bioink design and customization plays
methacryloyl-modification degree, polymer concentration, and a critical role in printability, cell viability and function, and the
UV exposure time.58,103,214 The crosslinking density and modulus of physical properties of the resulting tissue constructs. Natural
GelMA hydrogels increases with higher GelMA concentration, longer and synthetic polymer biomaterials are physically or chemically
curing time, and higher degree of methacryloyl-modification. crosslinked to form 3D networks that provide an ECM-like matrix
Usually, single-component hydrogels show limited tunability in to supporting cell growth. Bioinks composition and crosslinking
mechanical properties. To this end, multicomponent biomaterial methods can be customized to tune printability. Biorthogonal
hydrogels, including hybrid hydrogels, nanocomposite hydrogels, crosslinking approaches that use precursors and by-products with
and double-network hydrogels, with tunable mechanical properties low toxicity, suitable crosslinking kinetics, and compatibility with
and enhanced toughness, are developed.13,34,215 For instance, Rutz physiological conditions are preferred in bioink design. Among
et al. reported a PEGX toolkit to manipulate the properties of these, the rapid ionic crosslinking of polysaccharides is widely used.
PEG-based bioinks.185 PEG is functionalized with reactive groups Chain-growth photopolymerization and click chemistry-based
(represent the “X”) on both ends and act as a crosslinker for crosslinking methods are extensively used to form biomaterial
various polymers. The chain length and architecture (linear or hydrogels from polysaccharides and protein derivatives with good
multi-arms) of PEGX crosslinker can be altered. Linear (e.g., physiological and environmental stability. Bioactivity and network
gelatin), branched (e.g., four-arm PEG amine), or multifunctional topological structures of crosslinked biomaterial hydrogels are
(e.g., GelMA) polymer can be mixed with PEGX and cells modulated to enhance cell behavior. The mechanical properties
to formulate the bioinks. The use of thiol Michael addition of biomaterial hydrogels are engineered using multicomponent
and tetrazine–norbornene click chemistry allows for good cell bioinks with multiple crosslinking mechanisms, including
viability.184 The mechanical properties of the resulting gel can be copolymerization of hybrid hydrogels nanocomposite hydrogels,
tuned depending on the concentration of PEGX and its molecular and DN hydrogels. Recently, many more natural and synthetic
weight and functional groups. Moreover, secondary crosslinking polymers have come into commercialization as bioinks. The wider
can increase mechanical robustness after printing. Besides, use of these materials, and continuous innovation in bioink design
various nanoparticles, including gold nanorods, laponite, and will broaden bioprinting research and deepen its impact in tissue
hydroxyapatite, can also be used to make composite GelMA bioinks engineering and regenerative medicine.
16 Design of Bioinks for Bioprinting

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20 3D Bioprinting of Functional Tissue Models

3D Bioprinting of Functional
Tissue Models

Min Tang,1 Shangting You,1 Jennifer Sun,2 Wei Zhu,2 Shaochen Chen1*
1
Department of NanoEngineering, University of California, San Diego,
9500 Gilman Drive, La Jolla, California, 92093, USA.
2
Allegro 3D, Inc, 6868 Nancy Ridge Drive, San Diego, California, 92121, USA.
*
Email: [email protected]

Introduction
The emergence of new technologies in tissue engineering and Inkjet-based Extrusion-based Digital light processing
Thermal Piezoelectric Mechanical Pneumatic
regenerative medicine promises to make a number of previously
impossible applications a reality, such as personalized functional Digital
micromirror
human tissues and organ replacement. Among currently available device (DMD)
Light chip
strategies and techniques, 3D bioprinting is creating a paradigm Heater Actuator
shift in how to build a wide range of complex tissues that Vapor
bubble Polymerized
recapitulate the in vivo cellular heterogeneity, microarchitectures, patterns

biophysical properties, and biochemical constituents. More Uncured

bioink
specifically, 3D bioprinting enables precise control over the
patterning of cells and biomaterials to mimic and ultimately
Figure 1. Different modes of 3D bioprinting technologies. Adapted with
replace the native arrangement of tissues. To date, numerous permission from reference 75, copyright 2021 Wiley.
tissues have been fabricated using 3D bioprinting technology,
including but not limited to liver, heart, lung, kidney, blood
vessels, cartilage, and placenta.1–7 These bioprinted tissues Inkjet-based bioprinting
have essential applications in various biological and medical The 3D inkjet printer, first adopted from common desktop ink-
250
areas, serving as physiologically relevant models to elucidate based paper printers, deposits biomaterials and cells in a raster-
biological mechanisms and screen drug compounds, lab-on-chip like manner.10 Inkjet-based printing methods include continuous
devices when integrated with biosensors and microfluidics, or inkjet printing and drop-on-demand inkjet printing. In continuous
transplantable tissues and regenerative medicines. This article inkjet printing,11 droplets are formed by the capillary-driven
reviews the current state of 3D bioprinting, highlights current Rayleigh-Plateau instability of the liquid column. Then, these
work on 3D bioprinted tissue models, and discusses advanced droplets are charged and can be deflected by an electric field,
bioinks such as gelatin methacrylate (GelMA) and decellularized allowing them to be controlled to either deposit on the build
extracellular matrix (dECM) that can facilitate the development of platform or enter a recycling collector. In drop-on-demand inkjet
physiologically-relevant tissues. printing,12 droplets are only generated when the inkjet head is
aligned with the desired area, directly depositing on the build
3D Bioprinting Technologies platform. The deposited bioink is then thermally, photochemically,
Several different 3D bioprinting technologies (Figure 1) are or ionic-interactionally crosslinked. These printers are capable of
currently capable of processing cell-laden biomaterials and ~50 µm typical printing resolutions but can only be used with low
directly constructing complex 3D structures. These technologies viscosity materials.13,14
include inkjet-based 3D printing, light-based 3D printing, and
extrusion-based 3D printing, with each varying in terms of print
resolution, speed, and biocompatibility.8,9
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 21

Extrusion-based bioprinting Bioinks

Extrusion-based bioprinters are based on the widely used fused 3D bioprinting techniques now utilize a growing variety of natural
deposition modeling (FDM) 3D printers.15 Generally, biomaterials biomaterials and synthetic materials for biomedical applications.
are extruded through a nozzle tip using pneumatic or mechanical Natural materials, such as gelatin, hyaluronic acid, collagen, and
pressure and deposited on the build platform in a raster-like derivatives, possess good biocompatibility and innate bioactive
manner. The deposited bioink is then thermally, photochemically, features that are ideal for cell growth. Synthetic materials lack the
or ionic-interactionally crosslinked. These printers are relatively inherent biochemical cues of natural materials but have tunable
low cost, have ~50 µm typical resolution, and can accommodate mechanical properties suitable for bioprinting purposes. Composite
a larger range of material viscosities than inkjet printers.16,17 materials or chemical modifications to materials are standard
Extrusion-based 3D bioprinting technique are capable of high methods that improve the biomimicry or printability of bioinks.
cell density or cell spheroid printing.18 However, the shearing
force applied to the extruded biomaterials during extrusion can Gelatin and GelMA
decrease cell viability.8 Gelatin, a partial hydrolysis product of collagen, is widely used in
extrusion-based bioprinting due to its good rheological properties
Light-based bioprinting and thermal responsiveness. It possesses good biocompatibility
The concept of light-based 3D printing is inspired by and intrinsic bioactivity, including integrin-binding and MMP
photolithography. The liquid state printing materials can be digestion sites. Gelatin is used for many tissue printing models,
photochemically polymerized and solidified upon light exposure; including skin tissues and liver tissues, due to the presence
thus, a 3D structure is made by selectively delivering light of collagen in many organs. Hepatocytes encapsulated in
energy to the desired location. Light can be delivered via 3D-bioprinted gelatin hydrogel can remain functional over two
scanning fashion, such as in stereolithography,19 or through months of culture.30 When mixed with synthetic materials to
imaging projection such as DLP-based bioprinting.20,21 Scanning form composite bioinks, gelatin-based biomaterials exhibit higher
stereolithography induces photopolymerization at the focal resolution and printability while maintaining cell viability and
spot of the focusing light and creates 3D structures by proliferation.31 GelMA is one of the most widely used biomaterials
scanning. Microstructures can be produced with a micron-scale for light-based bioprinting due to its photo-sensitivity and gelatin-
resolution;19,22 even sub-micron scale resolution is possible when derived biological features. GelMA is synthesized from gelatin by
a two-photon photopolymerization is used.23,24 substituting the lysine and hydroxyl groups with methacrylamide
and methacrylate side groups;32 the rate of substitution impacts the
DLP-based 3D bioprinting has emerged as a promising
mechanical properties of the bioprinted GelMA constructs. Various
technique. Unlike inkjet-based, extrusion-based, or scanning
tissue models such as liver models, heart models, conjunctiva
stereolithography, where construction of a 3D structure occurs
models, as well as disease models, including glioblastoma models
in a point scanning method, DLP-based 3D printing projects a
and liver cirrhosis, have been developed using GelMA.33–36 GelMA
2D pattern and polymerizes the entire plane simultaneously,
has also been combined with synthetic materials such as PEGDA
achieving much faster printing speed. In general, the 3D model
to generate a cardiac patch to treat myocardial infarction or
is sliced into a series of 2D cross-sections and projected on the
regenerative construct for spinal cord injury.37,38
printing material to polymerize one layer of the 3D structure. The
motorized stage controls the vertical position of the printed part GMHA, HAMA
and allows printing of the subsequent layer in a discrete layer-by-
Hyaluronic acid (HA) is another ubiquitous extracellular matrix
layer fashion25,26 or a continuous layer-less fashion.27,28 The typical
(ECM) component. It is comprised of linear polysaccharides
fabrication resolution of DLP-based 3D bioprinting is a few microns
with alternating d-glucuronic acid and N-acetyl-d-glucosamine.39
where a de-magnification lens is used.28
The negatively charged HA can attract a large volume of water,
Compared to inkjet-based and extrusion-based 3D printing, making it ideal for regulating the hydration state, porosity, and
light-based 3D printing can achieve better fabrication resolution various other mechanical properties of many tissues. HA also
(micron and sub-micron scale) due to the precise manipulation of plays a critical role in regulating physiological processes and
light. Also, DLP-based 3D printing can achieve a printing speed is involved in various pathological events through interaction
that is a thousand times faster than an extrusion-based printing with cells and other ECM components. Unmodified HA lacks
method. Furthermore, light-based 3D printing has demonstrated good printability, but it can be combined with various printable
suitability for cell encapsulation to form biomimetic tissue biomaterials to generate 3D tissue models for mechanistic studies
constructs1,5,27,29 where higher cell viability is achieved as the and phenotypic analyses.40–43 For example, bioprinted brain
shearing force or heating is absent. However, the light-based tumor models demonstrate higher invasiveness in HA-GelMA
method requires photopolymerizable materials, which limits the hydrogel consisting of low molecular weight HAs than in high
range of material choices. molecular weight HA models.41 HA-based biomaterials with
different molecular weights are appropriate for modeling brain
To date, new bioprinting techniques with improved performance tissue and many tumor tissues.44 Chemical modifications to
and novel features are being actively researched and developed. improve printability of HA generally target the carboxylate group,
Each technique has its advantages and disadvantages, and it is the N-acetyl group, or the hydroxyl groups on both moieties.
essential to select the most suitable bioprinter for the specific
bioapplication carefully.
22 3D Bioprinting of Functional Tissue Models

Glycidyl methacrylate functionalized HA (GMHA), or methacrylic PEGDA

anhydride functionalized HA (HAMA) are popular bioinks for light- Various synthetic materials have been explored for 3D printing
based bioprinting, especially DLP-based bioprinting, due to their purposes. Polyethylene glycol (PEG) is a biocompatible and bio-
photo-responsiveness once chemically modified. Brain tumor inert synthetic polymer that can be combined with other bioinks
models and liver tissue models have been developed with GMHA or peptides to improve biomimicry for tissue modeling.56–58 Brain
using DLP-based bioprinting techniques.33,34 Unlike gelatin-based tumor models fabricated with PEG-HA hydrogel functionalized with
biomaterials, HA-based materials do not possess innate Arg- RGD peptides and MMP degradation crosslinkers display different
Gly-Asp (RGD) sites; thus, HAMA functionalized RGD peptides cell morphologies at different stiffness states.59 A derivative of
have been bioprinted to facilitate cell adhesion.45 HA-based PEG, polyethylene glycol diacrylate (PEGDA), is light-responsive
constructs can also be developed using extrusion-based or inkjet- material suitable for light-based printing methods with good
based bioprinting techniques through addition and condensation biocompatibility and tunable mechanical properties.38 PEGDA
reactions. For example, HA-thiol biomaterials can spontaneously combined with GelMA has been used to fabricate regenerative
polymerize through the formation of disulfide bonds.46 constructs through DLP-based bioprinting for spinal cord injury,
exhibiting good biocompatibility and long-term integrity when
dECM
implanted.60,61
dECM has emerged as a popular next-generation bioink due to
its ability to preserve the complexity of native ECM composition. 3D Bioprinted Functional Healthy/Diseased
The biochemical cues of dECM can support tissue-specific cell Tissue Models
growth and behaviors.36 After removing cellular components,
Multiple cell types co-exist in vivo and communicate with each
dECM derived from patient brain tissue shows that significant ECM
other through paracrine signaling and interactions with the
components, including glycosaminoglycans, HA, collagen, laminin,
surrounding ECM microenvironment to form a functional tissue
and fibronectin, are preserved.47 Similarly, dECM derived from
unit.62 Creating truly biomimetic tissues in vitro requires the
liver tissue shows native ECM components such as collagen I,
recapitulation of heterogeneous cell populations and their proper
collagen IV, laminin, and fibronectin.36 DECM is often mixed with
spatial arrangement within a relevant ECM microenvironment. As
other printable biomaterials for tissue modeling for bioprinting
such, 3D bioprinting technologies offer a precise way to pattern
purposes due to its relatively slow gelation kinetics. Recently,
various cell types and biomaterials. The source of cells is also
dECM has also been independently printed through thermal
of great importance in the context of creating physiologically
gelation.48–50 Liver dECM mixed with GelMA has been used to
relevant tissue models. The majority of printed tissues produced
fabricate the liver cancer model through DLP-based bioprinting
to date use established cell lines or animal cells, but these cells
with different stiffness matching the healthy and cirrhosis
cannot fully reflect the behavior and function of human primary
states.36 Patient brain dECM mixed with collagen has been used
cells.63 Human stem cells (hSCs), including embryonic stem cells
to generate brain cancer models with improved printability on
(hESCs) and induced pluripotent stem cells (hiPSCs), with the
extrusion-based bioprinters.47 dECM enhanced the heterogeneity
proliferation capacity and potential to differentiate into various cell
of tumor cell morphology and the expression of matrix remodeling
types such as cardiac cells, liver cells, and neuronal cells, have
protein tumor cells compared to a pure collagen hydrogel. Various
promising applications in 3D bioprinting.64 Primary human cells
other tissues, including heart, adipose, and cartilage, have been
derived from healthy individuals or patients are also desirable for
bioprinted using dECM-based bioinks.36,49–52 Despite the potential
biomimetic in vitro tissue modeling.
downside of batch variation due to the diversity present from
its primary sources, dECM has opened an avenue of precision Biomimetic Human Healthy Tissue Models Based on
medicine applications. hSCs
Diseases associated with liver and heart are significant
Alginate
contributors to mortality in the United States.65,66 Additionally, the
Alginates are derivatives of alginic acid from brown algae. Alginate
liver is involved in xenobiotic and drug metabolism, making the
bioinks are commonly employed in extrusion-based bioprinting
investigation of hepatotoxicity essential to drug studies. Another
because of their adept gelation and their good mechanical
critical factor for evaluating novel drugs is cardiotoxicity, which
properties. Various chemical modifications to alginate, such as
is the primary reason for drug retraction from the market.67
methacrylation and norbornene functionalization, enable their
Various 3D bioprinting approaches combined with human iPSCs
use in light-based bioprinting or improve their polymerization and
have been explored to create in vitro liver or cardiac tissues for
degradation kinetics.53,54 Alginate hydrogels have demonstrated
use in these applications.
good biocompatibility and have been used to develop different
tissue engineering applications and tissue types, such as cardiac Human iPSC and ESC-derived hepatocyte-like cells have been
tissue, cornea, bone, and cartilage.55 encapsulated by inkjet-based bioprinting in alginate hydrogels
to create 3D liver constructs.68 Hepatic cells in these 3D-printed
constructs showed good viability and functionality such as
albumin secretion. A more complex and biomimetic hepatic
model has been developed using DLP-based bioprinting, with the
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 23

encapsulation of hiPSC-derived hepatic progenitor cells (hiPSC- extrusion-based approach encapsulated iPSC-CMs and HUVECs in
HPCs), human umbilical vascular endothelial cells (HUVECs), and alginate and PEG-fibrinogen hydrogel to produce an implantable,
human adipose-derived stem cells (ADSCs). GelMA was mixed pre-vascularized cardiac patch.71 Patient-derived iPSCs can be
with hiPSC-HPCs for the hepatic region, and GelMA-GMHA was differentiated into iPSC-CM and iPSC-derived endothelial cells
mixed with HUVECs and ADSCs for the vascular region. Patterning as cell sources for a personalized cardiac patch fabricated by
of the hepatic and vascular regions via a two-step printing process extrusion-based bioprinting.72 A DLP-based bioprinting approach
mimicked tissue-scale anatomical features and dimensions of the has been used to generate cardiac arrays for high throughput
actual liver (Figure 2). drug screening.35 Different orientations of iPSC-CMs were printed
on force gauge arrays, and the iPSC-CMs exhibited the highest
Immunohistochemical staining of albumin and E-cadherin in
expression of cardiac-related markers and enhanced contractile
3D hepatic tri-culture constructs revealed more extensive
forces under drug treatment. A recent study used freeform
aggregation and spheroid formation than hiPSC-HPC only 3D
reversible embedding of suspended hydrogels (FRESH), an
controls, suggesting a higher degree of cell junction formation,
extrusion-based technique, to fabricate human heart tissues
maturation, and functionality. Tri-cultured hepatic cells also
using hESC-derived cardiomyocytes (hESC-CM) and chemically
expressed a higher level of crucial liver markers, including
unmodified collagen (Figure 3A).73 Spontaneous contraction of the
albumin, hepatocyte nuclear factor 4 alpha (HNFa), transthyretin
bioprinted cardiac ventricle was observed after 4 days of culture,
(TTR), and alpha fetal protein (AFP), compared to the 3D
and synchronous wave propagation was confirmed after 7 days
hiPSC-HPC only control and the 2D monolayer control. The 3D
(Figure 3B,C). Mechanical integrity of the collagen constructs was
hepatic tri-culture model could also serve as a platform for drug
confirmed using a tri-leaflet heart valve model. Lastly, a neonatal-
screening. Assessment of anabolic and catabolic functions of
scale heart consisting of internal structures such as trabeculae
hepatic cells in 3D printed constructs occurred by examining
and blood vessels was printed using this technique as a proof-of-
the expression of crucial cytochrome P450 (CYP) enzymes
concept for full-scale heart printing (Figure 3D).
involved in liver drug metabolism.69 Baseline CYP levels without
drug treatment showed that the 3D tri-culture model expressed 3D bioprinting can produce functional hepatic and cardiac models
elevated levels of CYP3A4, CYP1A2, CYP2B6, CYP2C9, and that recapitulate both the native tissue architecture and cellular
CYP2C19 compared to the 3D hiPSC-HPC only and 2D control. diversity. Incorporating hSC-derived cells in the tissue models
Upon introducing rifampicin, a known bactericidal antibiotic drug enables their potential applications as patient-specific models to
to induce hepatotoxicity,70 the 3D tri-culture model exhibited study various pathophysiological disease mechanisms and serve
increased CYP3A4, CYP2C9, and CYP2C19 expression, while no as drug screening and discovery platforms.
significant change in CYP expressions was observed in the 3D
hiPSC-HPC only or 2D controls. Biomimetic Human Diseased Tissue Model
In addition to healthy tissue models, patient-derived disease
Cardiac tissues have also been bioprinted using iPSC-
models and cancer models are essential tools for investigating
derived cardiomyocytes (iPSC-CMs) and various bioinks. An
disease mechanisms and drug development. Due to the flexibility

A) B) C)
Digital micromirror 2. Collagen Cell ink
0.9 mm 0.45 mm
device chip
1. 4.8 mm
Sequential input
of digital masks
8 mm
0.3 mm
Support Print
material
2 mm

D)
UV Light
(365nm)
Sequential input of
Projection cell-material solutions
lens
55 mm

Coverslip
2. Liver lobule
analog
1.
Right Left
ventricle ventricle
37 mm 5 mm

Figure 3. Human cardiac constructs by FRESH 3D bioprinting.

A) Schematic of dual-material FRESH printing. B) The ventricle model has
Figure 2. Human hepatic constructs by DLP-based 3D bioprinting. The a central section of cardiac cells (pink) and a collagen shell (green and
construct was fabricated through a two-step printing process in which yellow). C) Image of the bioprinted ventricle. D) Neonatal-scale human
hiPSC-HPCs were patterned as hexagonal shapes (green) first, followed by heart printed based on a human heart MRI scan using FRESH. Adapted with
the patterning of supporting cells (red): scale bars, 500 μm. Adapted with permission from reference 73, copyright 2019 Springer.
permission from reference 1, copyright 2016 National Academy of Sciences.

250
24 3D Bioprinting of Functional Tissue Models

and customizability of 3D bioprinting technology, it is especially demonstrated enhanced invasion and resistance to chemotherapy,
suitable for precision medicine cancer models and recapitulation while macrophages in the tetra-cellular model exhibited
of the tumor heterogeneity to facilitate novel drug development. spontaneous polarization towards pro-tumor M2 phenotype. The
model could be used as a drug screening platform to predict
A regionally patterned liver cancer model has been developed
therapeutic outcomes or a CRISPR-Cas9 screening platform to
using a DLP-based bioprinter with liver dECM, collagen, GelMA,
identify novel drug targets (Figure 4D). Genes identified as
and HepG2 liver cancer cells.36 Here, the stiffness of the printed
essential in 3D-printed models were critical to cell viability in vitro
hydrogel was tuned to mimic the stiffness of liver cirrhosis and
and involved in a shorter survival time of experimental animals.
normal tissue, while the dECM provided biomimetic ECM cues
for cell growth. It was observed that HepG2 cells demonstrated Conclusions
enhanced invasion markers in the liver cirrhosis model compared
3D bioprinting technology has led to many breakthrough
to healthy controls. The work demonstrated the potential of DLP
developments of in vitro tissue models and disease models
printing in recapitulating specific mechanical properties of native
for applications in mechanistic studies, drug screening, tissue
tissue while preserving the biochemical feature using natural
engineering, and regenerative medicine. DLP-based 3D
biomaterials.
bioprinting enables rapid fabrication of complex cell-encapsulated
Brain tumor models have also been generated using different architecture with microscale resolution and physiologically
bioprinting techniques. An extrusion-based bioprinting method relevant structures. The human biomimetic 3D hepatic triculture
utilized brain dECM, patient-derived glioblastoma cells, and and cardiac models developed by DLP-based printing composed
endothelial cells to investigate patient-specific responses to of human iPSC-derived hepatic progenitor cells or iPSC-derived
chemotherapy (Figure 4A).74 The glioblastoma-on-a-chip consisted cardiac cells have enhanced functionality compared to traditional
of a tumor region, an endothelial region, an empty chamber for culture methods. They can provide reliable evaluation of drugs
medium, and a silicon wall to create oxygen gradient in the and compounds. Extrusion-based printing has the advantage of
tissue. Various drug combinations were tested using a bioprinted simplified scale-up to the size of the printed constructs using
chip to identify treatment plans with superior tumor killing unmodified natural materials, but with relatively limited printing
ability. A multicellular glioblastoma model was also developed resolution compared to light-based printing. In conclusion,
using DLP-based bioprinting and GelMA and GMHA as bioinks the future direction of 3D bioprinting for in vitro tissues and
to encapsulate patient-derived glioblastoma stem cells (GSCs), organs will involve multiple facets, namely, heterogeneous
macrophages, astrocytes, and neural progenitor cells (NPCs) in cell populations, proper biomaterials, relevant architecture
a tumor-parenchyma structure (Figure 4B).34 Macrophages were mimicking native structures, as well as intricate vascular and
co-printed with GSCs to form the tumor core, which mimicked neuronal networks to maintain long term growth and maturation
the actual cellular composition of glioblastoma tumor mass of the tissues, to provide a tissue-specific microenvironment for
(Figure 3C). Astrocytes and NPCs were printed on the periphery enhanced functionality and biomimicry.
to encompass the tumor core. GSCs in the tetra-cellular model

A) Bioink II
Glass substrate B) GSCs Macrophages
BdECM +
Bioink I GBM cells
BdECM +
Endothelial cells Astrocytes MMP site
RGD NSCs
c
Silicone Ink
Gas-permeable

Glycidyl methacrylates-HA
Glass substrate Inlet
Gelatin methacrylate

In Vitro 3D Glioblastoma Model

C) D) 1.5
Cancer Cell Line Encyclopedia
Brian Cancer Only
Bright Field GSC Macrophage DAPI Merge
Correlation with AUC

High Signature Score

1.0
= Resistant to Drug
dinaciclib
Tetra-culture

3-CI-AHPC
0.5 alvocidib
belinostat
BRD-A86708339
0.0

-0.5 ML334
High Signature Score
= Sensitive to Drug
-1.0
Ranked Therapeutic Compounds

Figure 4. Human glioblastoma models by 3D bioprinting. A) Glioblastoma-on-a-chip using extrusion-based bioprinting. B) Illustration of components of
a multicellular glioblastoma model using DLP-based bioprinting. Figures A and B adapted with permission from reference 74, copyright 2019 Springer. C)
Image of the bioprinted glioblastoma model. GSC (green), macrophage (red), NPC and astrocyte (blue). Scale bar, 500 μm. D) Prediction of drug sensitivity
based on the transcriptional profile of GSCs in the 3D bioprinted model. Figures C and D adapted with permission from reference 34, copyright 2020 Nature.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 25

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26 Hybrid printing for Engineering Vascularized Tissue Constructs

Hybrid printing for Engineering

Vascularized Tissue Constructs

Jiannan Li,1 Sungwoo Kim,1 Carolyn Kim,1,2 Yunzhi Peter Yang1,3,4*

1
Department of Orthopaedic Surgery, Stanford University, 240 Pasteur Drive, Stanford, CA 94304, USA
2
Department of Mechanical Engineering, Stanford University, 440 Escondido Mall, Stanford, CA 94305, USA
3
Department of Materials Science and Engineering, Stanford University, 496 Lomita Mall, Stanford, CA 94305, USA
4
Department of Bioengineering, Stanford University, 443 Via Ortega, Stanford, CA 94305, USA
*
E-mail: [email protected]

Introduction
The development of three-dimensional (3D) printing1 provides a printing mechanism can engineer multiple materials with the
powerful tool for tissue engineering, due to its unprecedented required distinct properties. Therefore, we envision integrating
capability to reproduce the spatial features of native tissues by multiple printing mechanisms, to engineer the desired complex
printing cell-laden constructs.2 However, vascularization arguably multi-cell, multi-material tissue constructs as the most promising
remains the most difficult yet most crucial challenge in tissue solution. This is referred to as hybrid printing or Hybprinting for
engineering, even with 3D printing techniques. To engineer large short. By integrating different printing mechanisms, Hybprinting
and complex functioning tissue constructs, the development of provides unprecedented coverage of material range and thus
proper vascular networks is required to ensure every individual the flexibility and capability to engineer complex, customizable
cell is within 100–300 μm from the nearest capillary to avoid cell biomimicking vascularized scaffolds. This review focuses on
death.3 Despite numerous breakthroughs to significantly improve using the Hybprinting approach for complex tissue engineering.
the resolution of 3D printing,4 the current printing resolution Specifically, in our envisioned strategy for engineering vascularized
is still not good enough to reconstruct the required capillary construct, either FDM or MME can be used to generate structural
networks, many of which have features smaller than 100 μm.5 support and provide the desired mechanical properties for the
More importantly, the engineering of thick vascularized tissue targeting tissue, SLA is used to print sophisticated patterns and to
is far more complicated than simply reconstructing a vascular generate major vessel branches of vascular tree complexity. Last,
network. It usually involves spatially controlled distribution of syringe extrusion can be used to expand the selection of bioinks
multiple phenotypes of cells and different extracellular matrices for fabrication of cell-laden tissue-specific scaffolds.
with distinct properties. Additionally, to engineer a vascularized
The approach can be combined with the localized distribution of
musculoskeletal composite tissue, one needs to combine bone,
growth factors for guided angiogenesis, forming microvasculature
cartilage, muscle, and tendon, which all possess different cell
networks. We believe this approach shows great potential for
compositions, mechanical properties, and biological functionalities
engineering thick vascularized tissues (Figure 1). To break down the
with their own vascular networks. The engineering and seamless
approach, we introduce each single printing mechanism, including
integration of such multi-cell, multi-material tissue constructs
FDM, syringe extrusion, and SLA. Meanwhile, we briefly describe
remain a major challenge. Various 3D printing mechanisms have
their roles in the proposed Hybprinting strategy for vascularization
been developed to target specific material types, such as fused
and potential applications. We hope this provides insights into
deposition modeling (FDM) or molten material extrusion (MME)
novel approaches in bioprinted vascularized tissue constructs and
for rigid polymeric materials,6 and stereolithography (SLA) and
contributes to various tissue engineering applications.
syringe extrusion for soft hydrogel materials.7,8 However, no single
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 27

A) B) the throughput and the cost-efficiency has improved significantly.

Moreover, SLA printing resolution has also been enhanced by orders
of magnitude with the development of two-photon lithography or
multi-photon lithography techniques.19 SLA printing can now
C) D) achieve resolution of several micrometers and even reach the
sub-micron resolution. This capability to generate high-resolution
patterns makes SLA an excellent fit for printing sophisticated
vascular networks. For example, B. Huber et al. reported using SLA
Figure 1. Envisioned strategy for engineering vascularized tissues: A)
printing to fabricate photo-curable cytocompatible polyacrylate (PA)
FDM for generating structural support with rigid polymeric materials; material for the generation of vessels.20
B) SLA for printing major vessels with hydrogel materials; C) syringe
extrusion for printing cell-laden, tissue-specific scaffolds. Different colors
Nevertheless, the resolution of SLA printing is still not
could represent various growth factors or material compositions enabled
by different syringe printing nozzles; D) Microvascular network are then sufficient enough to reproduce microvascular networks, since
formed by guided angiogenesis. microvasculature dimensions are so small. In our envisioned
strategy for vascularized tissue printing, SLA is to be mainly used
FDM for Structural Supports for printing major vessels, while microvascular networks would
need to be generated through guided angiogenesis. By perfusing
FDM is one of the most extensively used 3D printing techniques.
through the major vessels and incorporating proper cells and
First developed in 1989, the FDM technique expanded rapidly
growth factors in the surrounding scaffold, vascular sprouting can
and quickly showed the potential to revolutionize the prototyping
be induced from the major vessels into surrounding cell-laden
and manufacturing industry. Due to its low cost and modular,
hydrogels to spontaneously anastomose with microvasculature
customizable design, it has been widely applied to many
within the surrounding cell-laden hydrogel to establish a
fields, including aerospace, automobile, art, and biomedical
perfusable vascular bed. Our group has implemented this
applications. 6,9–11
The base material, usually filament or pellets,
is first heated in a barrel to melting temperature in a standard 250
strategy by developing a dual hydrogel system that sequentially
integrates a slower degradable hydrogel for long term sustained
FDM process. The melted material is then extruded through a
perfusion with a faster degradable hydrogel for microvasculature.
hot nozzle. Once extruded, the material cools and solidifies.
The desired pattern can be formed by coordinating the extruded
360 this induced vascularization approach using
We demonstrated
a gelatin methacrylate/poly(ethylene glycol) dimethacrylate
material with a translation system. In this process, the materials
(GelMA/PEGDMA) dual-hydrogel system loaded with human
used are usually thermal plastic polymers, such as acrylonitrile
umbilical vein endothelial cells (HUVEC)21 (Figure 2). We expect
butadiene styrene (ABS), polycarbonates, polylactic acid (PLA), 450
that combining such a technique with SLA bioprinting will lead to
polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), etc.
better reproduction of sophisticated vascular networks.
Many of these materials are food-grade, non-toxic materials,
and some of them even show excellent biocompatibility and
Syringe Extrusion for Tissue-specific Scaffold
biodegradability, making them great candidates for tissue
constructs. For example, PLGA has already been approved by Syringe extrusion is one of the most cost-effective printing
the FDA for therapeutic purposes, while PLA and PCL have also techniques and has also been used extensively in bioprinting
been extensively used in engineering tissue scaffolds. Scaffolds and tissue engineering.22 A syringe extrusion process is
fabricated with these materials are usually mechanically strong straightforward; it includes loading a prepared bioink into a
and therefore they are widely used in bone tissue engineering. 12–14 syringe, then extruding it via a nozzle by applying pressure to
However, their application is not only limited to bone engineering, the bioink. The key to bioprinting with syringe extrusion lies
as the mechanical property of the scaffold can be manipulated by within the design of the bioink, as it must meet many criteria,
geometrical design. In Hybprinting, FDM serves as one module including good printability, biocompatibility while possessing the
for printing thermoplastic scaffolds that act as structural supports desired mechanical property. Many different bioink formulations
while maintaining proper porosity to allow for other components. have been developed using all kinds of base materials, such
as GelMA,23 alginate,24 and PEG,25 for example. These bioinks
SLA for Major Vessel Branches have demonstrated excellent cytocompatibility as they can be
used for cell encapsulation and long-term in vitro culture. Other
SLA printing is well-known in the field of manufacturing for its ultra-
advantages of syringe extrusion are its low-cost and simple setup,
high resolution. Using light-curable material, SLA printing forms
which allow for easy customization of multiple printer heads in a
the desired shape in a layer-by-layer format by controlling the light
single platform. This capability facilitates simultaneous co-printing
pattern for each layer.15,16 Traditional SLA printing suffered from low
of multiple bioinks, with each bioink specifically designed for
throughput and high cost due to discontinuity in between layers
different tissues by changing the material composition and growth
and the high expense of custom, high-resolution photomasks or
factors. This makes syringe extrusion optimal for printing cell-
laser scanners. However, with the advancement of digital light
laden, tissue-specific scaffolds. For example, syringe extrusion
processing (DLP)17 as well as controlled oxygen-permeable optics,18
28 Hybrid printing for Engineering Vascularized Tissue Constructs

A) PEG channel Dual hydrogel Perfusion Vascularization

PEG GelMA Perfusion HUVEC

B) 35
Soft GeIMA C) 120
D)100
30 Stiff GeIMA 100

In vitro degredation (%)

In vitro degredation (%)

Young’s Modulus (kPa)

PEGDMA Soft GelMA

25
PEGDMA 80
Stiff GelMA
80
PEGDMA (collagenase) Soft GelMA (collagenase)
20 60
60 Stiff GelMA (collagenase)

15 40
40

10
20 20

5
0 0
0 2 4 6 8 10 12 14 0 7 14
0 Time (Days) Days

E)
(i) (ii)

(iii)

(i) (ii) (iii)

10 µm 10 µm 10 µm

in vitro cell culture

(iv)

(v)
EC protrusion Neovascularization

100 µm 200 µm

Figure 2. Development and characterization of an in vitro co-culture model for angiogenesis and vascularization. A) The manufacturing process of
the dual hydrogel system; B) Compressive modulus; in vitro degradation of C) PEGDMA and D) GelMA; E) Representative SEM images and microvessel
formation in an in vitro model system. (i) GelMA, (ii) Interpenetrating network (IPN) between the PEGDMA channel and the GelMA, (iii) PEGDMA, (iv)
HUVEC protrusion into GelMA at day 1, (v) microvessel formation in the dual hydrogel system at two weeks.

250
has been used for printing tendon,26 cartilage,27,28 muscle,29 or Hybprinting for Engineering Vascularized
nerve30 tissues. Notably, syringe extrusion has also been used Tissue
to generate major vessels;31 however, syringe extrusion is less
Each of the aforementioned printing techniques has its unique
360
effective when printing a complex structure containing bifurcating
pros and cons and works for specific types of materials. We
or manifold branch transition and hanging structures. In the
envision a platform that integrates these techniques will contribute
future, syringe extrusion may be used to print sacrificial materials
that are later removed to form blood vessels. 450
significantly to the engineering of complex tissues, including thick
vascularized tissues. Such a platform can utilize the advantages
Bioink preparation is one of the most challenging steps of syringe of multiple printing methods, allow for automated fabrication
extrusion printing, as the bioink is usually a composition of of multi-material constructs with mechanical and biological
viscous materials that requires proper mixing. Various efforts have gradients, and better mimic native tissues. In 2015, our group
been made for optimizing the preparation of bioinks, including developed a prototype of this bioprinting platform that integrated
passive mixing31 or manual active mixing.32 Recently, our group FDM and SLA for the very first time (Figure 3). The platform
has developed an automated active mixing platform (AAMP), achieved seamless integration of rigid and soft scaffolds34 and
which allows for fast, cost-effective, precisely controlled mixing helped to pioneer the Hybprinting strategy for printing complex
and preparation of hydrogel bioinks.33 We believe the improved tissues. In the future, multiple FDM and syringe extrusion units
bioink preparation will have great potential and a broad range of can be integrated into a single Hybprinting platform under a single
applications for bioprinting and tissue engineering. algorithm or human-machine interface.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 29

Meanwhile, several groups have been working on integrating A) B)

FDM and syringe extrusion. For example, Malda and co-workers MME
Module
developed a printing system using FDM to generate rigid
supporting structures and syringe extrusion to print cell-laden Hybrid
Construct Prepolymer
hydrogels.35 The Cho group proposed similar ideas at almost Solution
the same time.36 Additionally, the Atala group used integrated Build
Platform
FDM and syringe extrusion printing to generate anatomical
scale bioscaffolds with the help of sacrificial materials.37 In the
future, more and more different printing techniques can be Build Vat
integrated, expanding the capability of Hybprinting, and allowing
for the fabrication of more complicated and sophisticated tissue
Figure 3. In-house-developed combinatory bioprinting technology: A)
constructs. For example, in our envisioned strategy, growth view of the Hybprinter machine, B) FDM module and SLA vat of Hybprinter
factors can be patterned with either multi-syringe extrusion or and a typical scaffold-hydrogel part during the built process.
the introduction of an inkjet printing mechanism, resulting in a
growth factor gradient in the engineered scaffold. According to
(8) You, F.; Eames, B.; Chen, X. Int. J. Mol. Sci. 2017, 18 (7), 1597.
250
our previous study, the graded growth factor distribution can be (9) Boparai, K.; Singh, R.; Singh, H. Rapid Prototyp. J. 2016, 22 (2), 281–
specifically designed for guided angiogenesis, which will lead to 299.
(10) Klippstein, H.; Sanchez, A.; Hassanin, H.; Zweiri, Y.; Seneviratne, L. Adv.
the formation of microvascular networks, achieving the last step Eng. Mater. 2018, 20 (2), 1700552.
of engineering thick vascularized tissues. (11) Liu, Z.; Wang, Y.; Wu, B.; Cui, C.; Guo, Y.; Yan, C. Int. J. Adv. Manuf.
Technol. 2019, 102 (9–12), 2877–2889.
(12) Zein, I.; Hutmacher, D.; Tan, K.; Teoh, S. Biomaterials 2002, 23 (4),
Conclusion 1169–1185.
(13) Chen, G.; Chen, N.; Wang, Q. Compos. Sci. Technol. 2019, 172, 17-28.
This mini review focuses on bioprinting strategies for engineering (14) Khodaei, M.; Amini, K.; Valanezhad, A. J. Wuhan Univ. Technol. Mater.
Sci. Ed. 2020, 35 (1), 248–251.
thick vascularized tissue constructs. We reviewed different
(15) Ge, Q.; Li, Z.; Wang, Z.; Kowsari, K.; Zhang, W.; He, X.; Zhou, J.; Fang,
printing mechanisms, including FDM, SLA, and syringe extrusion, N. Int. J. Extrem. Manuf. 2020, 2 (2), 022004.
(16) Deshmane, S.; Kendre, P.; Mahajan, H.; Jain, S. Drug Dev. Ind. Pharm.
and their applications in printing vasculatures. Due to the 2021, https://doi.org/10.1080/03639045.2021.1994990.
complexity in engineering multi-cell, multi-material vascularized (17) Maines, E.; Porwal, M.; Ellison, C.; Reineke, T. Green Chem. 2021, 23
(18), 6863–6897.
tissue constructs, we envision that future strategies will need to (18) Tumbleston, J.; Shirvanyants, D.; Ermoshkin, N.; Janusziewicz, R.;
integrate multiple printing mechanisms in a single platform to Johnson, A.; Kelly, D.; Chen, K.; Pinschmidt, R.; Rolland, J.; Ermoshkin,
A.; Samulski, E.; DeSimone, J. Science 2015, 347 (6228), 1349–1352.
fabricate different complex tissues in a single step. We reviewed (19) Park, S.; Yang, D.; Lee, K. Laser Photonics Rev. 2009, 3 (1-2), 1–11.
this strategy for the printing of thick vascularized tissues, where (20) Huber, B.; Engelhardt, S.; Meyer, W.; Kruger, H.; Wenz, A.; Schonhaar,
V.; Tovar, G.; Kluger, P.; Borchers, K. J. Funct. Biomater. 2016, 7 (2), 11.
FDM is used for printing structural supports, SLA for major vessels, (21) Kim, S.; Pan, C. C.; Yang, Y. P. Macromol. Biosci. 2020, 20 (10),
syringe extrusion for cell-laden tissue-specific scaffolds, and e2000204.
(22) Jang, T.; Jung, H.; Pan, H.; Han, W.; Chen, S.; Song, J. Int. J. Bioprinting
inkjet for guided angiogenesis to create microvascular networks. 2018, 4 (1), 126.
Moreover, other printing mechanisms may also be integrated to (23) Du, M.; Chen, B.; Meng, Q.; Liu, S.; Zheng, X.; Zhang, C.; Wang, H.; Li,
H.; Wang, N.; Dai, J. Biofabrication 2015, 7 (4), 044104.
further broaden the range of capabilities. We believe Hybprinting (24) Chung, J.; Naficy, S.; Yue, Z.; Kapsa, R.; Quigley, A.; Moulton, S.;
has shown great potential in engineering thick vascularized tissues Wallace, G. Biomater. Sci. 2013, 1 (7), 763–773.
(25) Gao, G.; Schilling, A.; Hubbell, K.; Yonezawa, T.; Truong, D.; Hong, Y.;
due to its superior capability for handling complex cases and will Dai, G.; Cui, X. Biotechnol. Lett. 2015, 37 (11), 2349–2355.
have a promising future in tissue engineering applications. (26) Jiang, X.; Wu, S.; Kuss, M.; Kong, Y.; Shi, W.; Streubel, P.; Li, T.; Duan,
B. Bioact. Mater. 2020, 5 (3), 636–643.
(27) Kundu, J.; Shim, J.; Jang, J.; Kim, S.; Cho, D. J. Tissue Eng. Regen. Med.
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(28) Izadifar, Z.; Chang, T.; Kulyk, W.; Chen, X.; Eames, B. Tissue Eng. Part
This research was partially funded through financial support from C Methods 2016, 22 (3), 173–188.
(29) Distler, T.; Solisito, A.; Schneidereit, D.; Friedrich, O.; Detsch, R.;
NIH grants U01AR069395, R01AR072613, and R01AR074458 from Boccaccini, A. Biofabrication 2020, 12 (4), 045005.
NIAMS, and DoD grant W81XWH-20-1-0343, the Stanford Woods (30) Gu, Q.; Tomaskovic-Crook, E.; Lozano, R.; Chen, Y.; Kapsa, R.; Zhou,
Q.; Wallace, G.; Crook, J. Adv. Healthc. Mater. 2016, 5 (12), 1429–1438.
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30 Recent Advances in 3D Biofabrication of Blood Vessels

Recent Advances in 3D
Biofabrication of Blood Vessels

Alessia Longoni,1 Tim BF Woodfield,2 Jelena Rnjak-Kovacina,3 Khoon S Lim1,2*

1
Light Activated Biomaterials (LAB) Group, University of Otago Christchurch, New Zealand
2
Christchurch Regenerative Medicine and Tissue Engineering (CReaTE) Group, University of Otago
Christchurch, New Zealand
3
Graduate School of Biomedical Engineering, University of New South Wales, Australia
*
Email: author: [email protected]

Introduction
The human vascular system consists of an intricate hierarchical
A) B)
network of blood vessels of different sizes, which operate in unison
to ensure gas and nutrient exchange throughout the body (Figure
1A). Large blood vessels (≥ 6 mm in diameter) and capillaries
(≤10 µm in diameter) represent the extremes of this hierarchical
tree and play different roles in ensuring tissue homeostasis.
The primary function of macro-vessels like arteries is to buffer
the pressure associated with the intermittent left ventricular
contraction and act as a conduit to deliver steady blood flow to the C)
peripheral areas of the body. On the other hand, micro-vessels
are responsible for the transmural exchange of gas, nutrients,
and cellular waste (Figure 1B, C). The different functions of these
blood vessels are also reflected in their anatomy. Specifically, Nutrients CO2 O2 Waste
Blood flow
there is a progressive decrease in the vessel diameter and wall
thickness when moving from macro-vessels to micro-vessels.
The cellular organization and structure of these vessels are
also different, including having a different number of layers of
vascular smooth muscle cells that surround the endothelium, with
Figure 1. Schematic overview of the human vascular tree and vascular
capillaries within the microcirculation composed only of a single network function. A) Schematic representation of the main vessels that
layer of endothelial cells and supporting pericytes.1 are part of the human cardiovascular system. B) Representation of the
microcapillary network, which connects arterioles to venules. C) Gas and
Considering the heterogeneity in structure and function of different nutrient exchange that occurs at the capillary-tissue interface. While oxygen 250
and nutrients are delivered by the arterioles into the capillaries, CO2 and
types of blood vessels, it is not surprising that recapitulating the metabolic waste products are collected and transported by the venules.
complexity of the vascular tree remains a significant challenge Reprinted with permission from reference 3, copyright 2019 Elsevier Ltd.

in the field of tissue engineering and regenerative medicine.

Currently, several synthetic grafts and decellularized matrices are
tissue analogs which require a healthy capillary supply to allow
available for the replacement of large blood vessels to reconstruct
the exchange of gases, nutrients, and fluids.1,3 The formation of
or bypass vascular occlusions and aneurysms.1,2 On the contrary,
necrotic regions within engineered constructs is one of the leading
there is an unmet clinical need for strategies to engineer small-
causes of poor survival and integration with the host tissue,
diameter blood vessels to address occlusion in coronary and
limiting their regenerative properties and clinical translation.3,4
peripheral arteries and for the clinical translation of engineered
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 31

Over the past decade, various fabrication strategies to incorporate different sacrificial template designs with precise control over the
perfusable hollow microchannels/vessel-like structures into 3D resolution (diameter in the range of 45–1000 µm) and architecture
scaffolds have been extensively studied, often using conventional (Figure 2A). Specifically, individual templates with varying
templating and molding approaches. These approaches are often channel diameter — largest channel (650 µm) to smallest channel
simplistic with limited spatial architecture and resolution. The rapid (150 µm) in a hierarchical design — were successfully fabricated
advancement in the biofabrication field has enabled the creation and embedded within cell-laden hydrogel constructs to mimic the
of more complex and sophisticated shapes that has vast potential bifurcating motifs present in native tissues where large channels
to recapitulate the multiscalar architecture of native vasculature. often bifurcate into smaller channels for efficient blood flow and
nutrient transport.9
Biofabrication is defined as “the automated generation of
biologically functional products with the structural organization Although the aforementioned studies are impressive and show
from living cells, bioactive molecules, biomaterials, cell aggregates proof-of-concept for vessel-like hollow channel fabrication,
such as micro-tissues, or hybrid cell-material constructs, through printing multi-scaled clinically relevant-sized heterogeneous
Bioprinting or Bioassembly and subsequent tissue maturation constructs remains a challenge. Hence, to prevent deformation or
processes.”5 In this context, bioinks are further defined as “a collapse of the printed physiologically relevant sized structures,
formulation of cells suitable for processing by an automated recent studies have investigated the extrusion of hydrogel-based
biofabrication technology that may also contain biologically active materials into support baths. Noor et al. showed the possibility
components and biomaterials.”6 Thus, bioprinting is considered of fabricating heart analogs (height = 20 mm; diameter = 14
a subset technique of biofabrication, which involves automated mm) with major blood vessels that are perfusable, by extruding
processes where living cells are directly printed within bioinks. cell-laden omentum hydrogels into an alginate/xanthan gum
Therefore, it is important to note that printing approaches where support bath (Figure 2B).10 More recently, Skylar-Scott et al.
structures are first printed then seeded with cells do not qualify as extruded gelatin-based sacrificial ink into a cell-laden supporting
bioprinting, as cells were not included during the printing process. bath containing iPSCs aggregates to engineer perfusable cardiac
embryoid bodies11 (Figure 2C). After removing the gelatin
This article outlines advances in biofabrication technologies used
sacrificial ink, the constructs were perfused with a hyper
to construct the distinct hierarchical levels of the vascular system
oxygenated medium (95% O2, 5% CO2) at a flow rate of 250 µm/
using seminal papers in the field. In addition, the extent to
min, which significantly improved cell viability and functionality.
which engineered vessels recapitulate the physiological function
The functional performances of these constructs were further
of native vessels, current applications of biofabricated vessels,
examined using perfusion of various cardiac-related drugs, where
challenges, and future directions in the field will be discussed.
perfusion of isoproterenol through the embedded channel resulted
in a doubling of the spontaneous beating frequency. In contrast,
Biofabrication Approaches in Engineering
when 1-heptanol was perfused through the channels, a reduction
Vasculature in the overall tissue contractile amplitude was observed.
In the past years, numerous technologies have been developed
in the biofabrication field (i.e., ink-jet printing, laser-induced Another study by Soliman et al. showed that incorporating
forward transfer, extrusion-based printing, and lithography-based sacrificial templates of different designs into cell-laden hydrogels
printing),7 which have been applied to tissue engineering and can affect cellular behavior.4 For example, sacrificial channels of
regenerative medicine fields. To date, both extrusion-based and different fiber orientations (30°, 60°, or 90° between adjacent
lithography-based biofabrication modalities are most commonly layers) affected microcapillary formation driven by endothelial
used to create vessel-like structures within 3D constructs. cells and stromal cells encapsulated in the surrounding hydrogel.
It should be noted that most of these studies are not bioprinting,
Extrusion-based biofabrication technology involves precise where cells are directly extruded with the sacrificial inks. However,
deposition of materials in cylindrical filaments onto a receiver as technology progresses, co-axial extrusion approaches are
platform, according to computer-driven spatial instructions, being explored for direct 3D bioprinting of vascular constructs.
allowing layer-by-layer formation of 3D constructs with predesigned Cui et al. reported on the fabrication of blood vessels (diameter
structures.1 Miller et al. demonstrated extrusion-printing of = 1 mm) with spatial localization of cells, where the inner
carbohydrate glass used as sacrificial templates in engineered chamber of the co-axial extrusion apparatus consists of
tissues containing living cells.8 In general, the ‘sacrificial ink’ is endothelial cells in a fugitive crosslinking slurry. In contrast, the
first deposited and embedded in a hydrogel matrix. The printed outer chamber is a catechol-functionalized gelatin-methacryloyl
template can be easily removed via dissolution in cell culture hydrogel containing smooth muscle cells.12 This approach mimics
media, leaving open perfusable channels (diameter = 200µm) the distinct cellular orientation and organization of native blood
that can be further lined with endothelial cells. The success vessels, where the inner layer is often an endothelium (single
of this technique led to the use of several other materials as layer of endothelial cells) surrounded by an outer layer consisting
sacrificial inks, such as poloxamer (Pluronic F127), gelatin, of smooth muscle cells.
and alginate. For example, Kolesky et al. showed extrusion of
32 Recent Advances in 3D Biofabrication of Blood Vessels

A)

1 mm 1 mm 2 mm

B) Printing in support Crosslinking in Extraction from Culture in

medium 37° C suppoirt medium growth medium

C) Right CAD model Front view side view perspective view

vent. Left
vent. Printing
septal branches

Sept.
Diag.
Top view
Filling with
cardiac organoid
matrix

Right Septum Left

ventricle LAD ventricle

Septal Diagnal
branches branches Printing
diagonal branches

Figure 2. A) Pluronic F127 was used as fugitive ink by Kolesky et al. to print perfusable tubular vessel structures of different dimensions or hierarchical
networks. Reproduced from reference 9 under terms of the CC-BY license, copyright 2014 John Wiley and Sons. B) Overview of the biofabrication
workflow, starting from a human heart CAD model to its printing in a support bath and the extraction of the final product. Red and blue dyes indicated
hollow chambers of the left and right ventricles, respectively. Reproduced with permission from 10 under the terms of the Creative Commons CC-BY-NC
License. C) Overview of the biofabrication workflow to produce a perfusable cardiac tissue using sacrificial writing into a supporting bath containing iPSCs
aggregates. Reprinted from reference 11, copyright The Authors, some rights reserved; exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 license
http://creativecommons.org/licenses/by-nc/4.0/.
250
Other common biofabrication technologies used to recapitulate lithography-based technologies allow significantly greater spatial
the vessel architecture are light-based 3D-printing modalities. resolutions (25–50μm) that cannot be achieved 360 via extrusion-based
Light-based crosslinking processes allow control over the material biofabrication approaches. Grigoryan et al. recently showed the
spatiotemporal reaction, which can be leveraged to fabricate fabrication of a vascularized alveolar model using DLP technology
3D structures of high resolution.7 Furthermore, light-based (Figure 3B). The construct (height = 10 mm) was printed using
chemistries are known to be highly efficient while yielding minimal poly(ethylene glycol)-diacrylate (PEGDA) hydrogels at a print
450
and non-toxic by-products, which is an essential requirement time of 1 hour and voxel resolution of 5 pixels.13 Humidified
for the manufacturing of cell-laden constructs. Among the light- oxygen gas was infused into the air sac, while deoxygenated
based biofabrication technologies, lithography-based techniques red blood cells were perfused at the blood vessel inlet. In this
have been the most popular for vascularization applications, study, intervascular oxygen transport between networks were
providing the highest accuracy and precision to pattern 3D successfully demonstrated by the collected red blood cells at
constructs spatially.7 This technology is specifically dependent on the outlet having significantly higher oxygen saturation levels.
the fundamental photo-polymerization principle — spatial control Furthermore, the DLP-printed PEGDA hydrogels could withstand
of light irradiation to facilitate the sol-gel transition of a photo- >10,000 ventilation cycles (at 24 kPa and a frequency of 0.5
cross-linkable material. Light can be written (stereolithography, Hz) over 6 hours. It is to be noted that although this model is
SLA) or projected (digital light processing, DLP) into a bath acellular, the channels contain both convex and concave regions
of photocrosslinkable material, crosslinking specific regions that are architecturally similar to native alveolar air sacs. A study
of the material onto a computer-driven build stage via light by Levato et al. employed DLP technology to recreate the human
exposure (Figure 3A).7 By precise control over the voxel sizes, cerebral arterial circle (Willis’ circle) (Figure 3C).14 Convoluted and
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 33

A) Platform
B) Aveolar model technology

deoxygenated
Deoxy C)
blood RBCs
air sac?? Hydrogel
Bioresin hydrogel
aveolar
air duct air sack

air duct
oxygenated
with tidal
blood ventilation
Lens
deoxygenated
blood

hydrogel
alv. aveolar
alv. air duct

airway
Digital atrium

micromirror alv. alv. concave,

convex
airway Oxy
device oxygenated RBCs
blood

Laser

D) Rotating vial
Branch model

D
DM tio
n
jec n Photosensitive
pro tio
t3 jec n resin
pro jec
tio
t2
pro
t1

Light dose (area units): 80 mJ/cm2

Printed object Volumetric absorbed energy: 8.59 mJ/cm3
Print time: 10.1 s

Figure 3. Vascular network fabricated with light-based biofabrication technologies. A) Schematic illustration of DLP technique that exploits light to control
the spatial organization of biomaterials and cells. Adapted with permission from reference 7, copyright 2020 American Chemical Society. B) Vascularized
alveolar model fabricated using DLP technology by Grigoryan et al. Republished with permission from reference 13, copyright 2019 The American
Association for the Advancement of Science. C) Anatomical replica of a blood vessel present in the human Willis circuit were printed to prove the DLP 250
printing capability of reproducing a convoluted and irregular vessel-like structure. Reproduced with permission from 14 under the terms of the Creative
Commons CC-BY-NC License. D) Schematic overview of the volumetric printing principle of operation and the printed blood vessel structure. The obtained
branches were perfused with blue-dextran. Adapted with permission from 15 under the terms of the Creative Commons CC-BY-NC License.
360

out-of-plane channel networks with irregular vessel-like channels sprouting and neo-angiogenesis.1,3 Thus, efforts have been made
were printed within gelatin-methacryloyl hydrogels, successfully to go beyond the initial feasibility studies of using biofabrication
recreating an anatomical replica of the Willis’ circle based on 3D
450
approaches to fabricate acellular constructs containing vessel-like
angiographic images. More recent breakthroughs to light-based structures to 3D bioprinting of cell-laden constructs that dictate
biofabrication technologies have led to computed axial lithography cell behavior and functionality.
(CAL) technology, where the principles of computed tomography
Selecting the appropriate cell type and the source is also crucial
process were adopted to produce an optical 3D dose distribution of
as they directly affect the overall graft biofunctionality and in vivo
light required to photo-crosslink a material by combining a series
performance, i.e., interaction with host tissue. So far, different
of 2D light patterns. Each image projection propagates through
types of endothelial cells have been used for vascularisation
the material from a different angle, where the superposition of
purposes. The most frequent are human umbilical vein endothelial
exposures from multiple angles results in sufficient 3D energy
cells (HUVECs) isolated from primary vein segments, as they
dose to facilitate sol-gel transition. The main advantage of this
have been proven suitable for fabricating vessels of multiple
technology is the print speed, where Rizzo et al. demonstrated the
calibers.1,4,16–18 In addition to HUVECs, human microvascular
successful fabrication of a branched perfusable vascular model (10
endothelial cells (HMVECs) are derived from the microvessels
mm) within 10 seconds (Figure 3D).15
of various tissues (e.g., adipose, liver, and cardiac tissues) have
emerged as a promising source for engineering capillaries.1
Functionality Requirements for Biofabricated
Ideally, these cells would be of autologous origin, as this would
Vessels
ensure immunological compatibility of the engineered vascularized
To ensure clinical translation of the developed vascularized construct and prevent its rejection. However, cell heterogenicity
constructs, it is essential that the engineered vessels also and limited expansion potential are shortcomings of using
recapitulate the physiological function of the native ones. primary cell sources.1 To overcome these limitations, current
While vessels of different calibers have specific characteristics approaches focus on evaluating the feasibility of using induced
and functions, some general considerations apply to macro- pluripotent stem cell-derived endothelial cells (iPSC-ECs). iPSC-
and micro-vasculature. Firstly, the engineered vessels should ECs are generated from reprogramming somatic cells, thus are of
possess perfusable lumens, which allow physiological blood flow autologous origin. Furthermore, they have proven to have a great
without the formation of thrombi or occlusions.1 Secondly, the expansion potential while retaining their capability to differentiate
blood vessels should possess adequate mechanical properties into almost any cell type.1,11 Nevertheless, the ability of these
to withstand the pulsatile physiological pressure present at the cells to fully recapitulate endothelial cells mature phenotype
different levels of the vascular tree without bursting or deforming.1 and vessels functionality is still under investigation. In addition
Finally, the engineered blood vessels should integrate with the to endothelial cells, the presence of other cell types is required
host vasculature once implanted, either through anastomosis or
34 Recent Advances in 3D Biofabrication of Blood Vessels

to support the maturation of the vascular network and regulate 3D constructs, we believe that these next generation bioprinting
blood vessel functions. Specifically looking at microcapillaries, strategies might hold the potential to recapitulate both the
pericytes can both stabilize and influence the permeability of the complex architecture and functionality of the vascular system.
vessels network, as well as modulate endothelial cells behavior
through paracrine signalling.1,4 References
(1) Fleischer, S.; Tavakol, D. N.; Vunjak-Novakovic, G. Adv. Funct. Mater.
2020, 30 (37), 1910811.
The field is rapidly progressing into the bioprinting space, where
(2) Kirkton, R. D.; Santiago-Maysonet, M.; Lawson, J. H.; Tente, W. E.; Dahl,
newer studies include the aforementioned vessel-forming cells S. L. M.; Niklason, L. E.; Prichard, H. L. Sci. Transl. Med. 2019, 11 (485),
eaau6934.
(endothelial and pericytes) in the printing process to facilitate (3) Lim, K. S.; Baptista, M.; Moon, S.; Woodfield, T. B. F.; Rnjak-Kovacina, J.
cell-mediated micro-capillary formation within 3D bioprinted Trends Biotechnol. 2019, 37 (11), 1189–1201.
(4) Soliman, B. G.; Major, G. S.; Atienza-Roca, P.; Murphy, C. A.; Longoni,
constructs. Several studies have shown the presence of a vascular A.; Alcala-Orozco, C. R.; Rnjak-Kovacina, J.; Gawlitta, D.; Woodfield, T.
lumen and the expression of lineage-specific markers after 3D B. F.; Lim, K. S. Adv. Healthc. Mater. 2021, 11 (2), e2101873.
(5) Groll, J.; Boland, T.; Blunk, T.; Burdick, J. A.; Cho, D. W.; Dalton, P. D.;
bioprinting of endothelial (e.g., CD31, von Willebrand factor, Derby, B.; Forgacs, G.; Li, Q.; Mironov, V. A.; et al. Biofabrication 2016,
and VE-cadherin) and supporting cells (e.g., α-SMA and smooth 8 (1), 013001.
(6) Groll, J.; Burdick, J. A.; Cho, D. W.; Derby, B.; Gelinsky, M.; Heilshorn,
muscles heavy chain).11,12,16 In addition, vessels functionality was S. C.; Jüngst, T.; Malda, J.; Mironov, V. A.; Nakayama, K.; et al.
evaluated in vitro by perfusing the printed networks, assessing Biofabrication 2018, 11 (1), 013001.
(7) Lim, K. S.; Galarraga, J. H.; Cui, X.; Lindberg, G. C. J.; Burdick, J. A.;
their anti-thrombogenic properties by testing platelet activation, Woodfield, T. B. F. Chem. Rev. 2020, 120 (19), 10662–10694.
and investigating the vessels permeability and sprouting.12,15–19 (8) Miller, J. S.; Stevens, K. R.; Yang, M. T.; Baker, B. M.; Nguyen, D.-H. T.;
Cohen, D. M.; Toro, E.; Chen, A. A.; Galie, P. A.; Yu, X.; et al. Nat. Mater.
Nevertheless, only a limited number of studies have investigated 2012, 11 (9), 768–774.
(9) Kolesky, D. B.; Truby, R. L.; Gladman, A. S.; Busbee, T. A.; Homan, K.
biocompatibility, long-term integration, and remodeling of the A.; Lewis, J. A. Adv. Mater. 2014, 26 (19), 3124–3130.
fabricated vessels in vivo.12,13,20 While this is understandable (10) Noor, N.; Shapira, A.; Edri, R.; Gal, I.; Wertheim, L.; Dvir, T. Adv. Sci.
2019, 6 (11), 1900344.
considering the rapid evolution and progress of biofabrication (11) Skylar-Scott Mark, A.; Uzel Sebastien, G. M.; Nam Lucy, L.; Ahrens John,
technologies and bioinks, collecting these data would pave the H.; Truby Ryan, L.; Damaraju, S.; Lewis Jennifer, A. Sci. Adv., 2019, 5
(9), eaaw2459.
way for the clinical translation of 3D biofabricated vascular (12) Cui, H.; Zhu, W.; Huang, Y.; Liu, C.; Yu, Z.-X.; Nowicki, M.; Miao,
networks. S.; Cheng, Y.; Zhou, X.; Lee, S.-J.; et al. Biofabrication 2019, 12 (1),
015004–015004.
(13) Grigoryan, B.; Paulsen, S. J.; Corbett, D. C.; Sazer, D. W.; Fortin, C. L.;
Conclusions and Future Directions Zaita, A. J.; Greenfield, P. T.; Calafat, N. J.; Gounley, J. P.; Ta, A. H.; et
al. Science 2019, 364 (6439), 458–464.
Currently, most vascular engineering strategies have focused (14) Levato, R.; Lim, K. S.; Li, W.; Asua, A. U.; Peña, L. B.; Wang, M.;
Falandt, M.; Bernal, P. N.; Gawlitta, D.; Zhang, Y. S.; et al. Materials
on fabricating a single compartment/level of the vascular tree Today Bio 2021, 12, 100162.
(macro- or micro-vessel only). Nevertheless, to ensure immediate (15) Rizzo, R.; Ruetsche, D.; Liu, H.; Zenobi-Wong, M. Adv. Mater. 2021, 33
(49), 2102900.
connection to the host vasculature and perfusion of the (16) Kolesky, D. B.; Homan, K. A.; Skylar-Scott, M. A.; Lewis, J. A. Proc. Natl.
engineered constructs, the recapitulation of vessels of different Acad. Sci. U. S. A. 2016, 113 (12), 3179–3184.
(17) Lee, V. K.; Lanzi, A. M.; Haygan, N.; Yoo, S. S.; Vincent, P. A.; Dai, G.
sizes and functions might be required. At this moment, although Cell. Mol. Bioeng. 2014, 7 (3), 460–472.
there have been numerous breakthroughs in biofabrication to (18) Song, K. H.; Highley, C. B.; Rouff, A.; Burdick, J. A. Adv. Funct. Mater.
2018, 28 (31), 1801331.
create the shape and architecture of a multi-scalar vasculature (19) Rider, P.; Voronov, E.; Dinarello, C. A.; Apte, R. N.; Cohen, I. J. Immunol.
network, the required functionality is still lacking. As the field 2017, 198 (4), 1395–1402.
(20) Zhu, W.; Qu, X.; Zhu, J.; Ma, X.; Patel, S.; Liu, J.; Wang, P.; Lai, C. S.;
is progressing from biofabrication to bioprinting technologies Gou, M.; Xu, Y.; Zhang, K.; Chen, S. Biomaterials 2017, 124, 106–115.
where the appropriate and essential cells are printed within the
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BUT THE
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Comprehensive biodegradable polymers
for biomedical research

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to achieve controlled and targeted delivery of
therapeutic agents (e.g. APIs, genetic material,
peptides, vaccines, and antibiotics).

We now offer the following classes of

high-purity biodegradable polymers:
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• End-functionalized biodegradable polymers

For more information, please visit:

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36 Bioprinting Protocol withReady-to-use TissueFab® Bioinks

Bioprinting Protocol with

Ready-to-use TissueFab Bioinks
®

Introduction Before you start: Important tips for optimal

TissueFab bioinks are ready-to-use bioinks formulated for high
® bioprinting results
cell viability and printability and are designed for extrusion- Optimize printing conditions. Optimize printing conditions (e.g.,
based 3D bioprinting. TissueFab® bioinks are biodegradable and nozzle diameter, printing speed, printing pressure, temperature,
compatible with human mesenchymal stem cells (hMSCs) and cell density) for the features of your 3D printer and for your
other diverse cell types. TissueFab® bioinks are compatible for application to ensure successful bioprinting. The suggestions
use with most extrusion-based bioprinters. TissueFab® bioinks below can guide you.
enable the precise fabrication of 3D cell models and tissue
Reduce bubble formation. If the bioink has air bubbles, the
constructs for research in 3D cell biology, tissue engineering,
bubbles may hamper bioprinting. Carefully handle the bioink
in vitro tissue models, and regenerative medicine.
when you mix and transfer to avoid bubble formation. Do not
vortex or shake vigorously.
Disclaimer
TissueFab® bioinks are for research use only, not suitable for Aseptic techniques. Follow standard aseptic handling
human, animal, or other use. Please consult the Safety Data techniques when preparing and printing the bioink and during
Sheet for hazard information and safe handling practices. cell culture.

Specifications Cell density. Resuspend the cell pellet to the appropriate

volume for the desired printed structure and cell density. Typical
Storage: Store TissueFab® bioinks at 2–8 °C. Protect from light
cell density for extrusion-based bioprinting is 1 to 5 × 10 6
by storing the bottle in a foil bag or wrapping in aluminum foil.
cells/ mL.
Stability: Refer to the expiration date on the batch-specific
Note:The number of prints obtained from each 10 mL bottle of
Certificate of Analysis.
bioink (a unit) varies depending on the printed structure. For
example, each 10 mL bottle contains enough material to print a
Materials
30 µL structure in each well of three 96-well plates or a 100 µL
Materials supplied structure in each well of four 24-well plates.
TissueFab® bioinks are supplied as follows:
Procedure
• 1 × 10 mL bottle (1 unit)
Bioprint
Materials required but not supplied
Cool the bioink filled printing cartridge to the appropriate
• Cultured cells (visit our website for an up-to-date list of temperature indicated in the printing parameters table below
cell types) using a “temperature-controlled printhead,” if available, or
• Appropriate cell culture medium place the cartridge in a 4 °C refrigerator for 10–15 minutes to
induce gelation.
• PBS (Cat. No. D8537)
• Sterile pipette tips for transferring bioink TissueFab recommended printing temperature and pressures
are listed in table 1. Please follow the printer guidelines as
• Sterile printing cartridge, piston, and nozzle/needle for
recommended by the manufacturer. Load the print cartridge
3D printing
onto the 3D printer and print directly onto a Petri dish or
• Extrusion-based 3D bioprinter multi-well plates. Adjust the flow rate according to the nozzle
• Water bath or incubator diameter, printing speed, printing pressure, and temperature.
• Micropipettes
• Light source
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 37

Table 1. Printing Parameters

Recommended Print Recommended Print

Product # Name
Temp (°C) Pressure (kPa)

905429 TissueFab® bioink (Gel)ma -UV/365 nm 15 60–70

918741 (Gel)ma -Vis/405nm 20 110–120

920983 (GelAlg)ma -UV/365 nm 19 60–80

921610 (GelAlg)ma -Vis/405 nm 19 70–90

906905 Sacrificial 25 50–60

919632 (GelHA)ma -UV/365 nm 18 80–100

919624 (GelHA)ma -Vis/405 nm 18 80–100

920975 (GelAlgHA)ma -UV/365 nm 17 50–70

922862 (GelAlgHA)ma -Vis/405 nm 20 60–70

905410 Alg(Gel)ma -UV/365 nm 20 70–80

906913 Alg(Gel)ma -Vis/525 nm 20 70–80

915033 TissueFab bioink Bone

®
Vis/405 nm 20 70–80

915025 UV/365 nm 20 70–80

915637 support gel 80 120–130

915963 TissueFab® bioink Conductive Vis/405 nm 20 70–80

915726 UV/365 nm 20 70–80

Troubleshooting
1. Bioink is incubated at 37°C for 30 minutes, but it is 3. Printed structure spreads and does not hold its shape.
still gel. Possible reasons: Bioink was diluted with cell culture
Possible reasons: Malfunction of the incubator; or the bioink medium that remained in the cell pellet; bioink was not
is crosslinked due to light exposure. cooled sufficiently before printing, or the printing pressure
was too high.
Solution: Ensure the temperature of the incubator/water
bath is correct, and make sure the bioink bottle is evenly Solution: Do not dilute the bioink. Make sure the bioink has
and adequately heated in the incubator/water bath. Do not been cooled according to the instructions before printing.
expose the bioink to light before printing. Adjust printing pressure to achieve sufficient flow of bioink.

2. Air bubble is trapped in the middle of the bioink in the 4. Interrupted flow or no flow during printing.
cartridge. Possible reasons: Insufficient printing pressure or nozzle is
Possible reasons: Air bubble was created during transferred partially or fully clogged.
or when cells were dispersed in the bioink.
Solution: Adjust the printing pressure to achieve a sufficient
Solution: Warm the cartridge at 37°C for 5–10 minutes or flow of bioink. If the problem persists, change the nozzle.
until the bioink becomes fluid. Turn the cartridge so the tip
faces up to allow any air bubbles to exit from the cartridge
tip. Gently tap the cartridge to help air bubbles pass through
the tip.
38 Bioinks Preparation

Bioinks Preparation
This section includes basic protocols for bioink preparation cell surfaces, resulting in the activation of specific signaling
for products from MilliporeSigma Partnerships; T&R Biofab, pathways. Gel stiffness or rigidity also affects cell migration
Advanced Biomatrix, and Rokit. differently in 3D versus 2D environments. Furthermore,
integrin-independent mechanical interactions resulting from the
Three-dimensional (3D) gels allow for the study of the effects
entanglement of matrix fibrils with cell extensions are possible in
of the mechanical properties of the extracellular matrix (ECM),
3D systems but not in 2D systems where the cells are attached
such as density and rigidity, on cell development, migration,
to a flat surface.
and morphology. Unlike 2D systems, 3D environments allow
cell extensions to simultaneously interact with integrins on all

PhotoCol™ Methacrylated Collagen

Bioink Preparation
Introduction
MilliporeSigma, in partnership with Advanced BioMatrix, offers Light Wave-
Product Cat. No. Photoinitiator Solution
PhotoCol™, a purified methacrylated Type I bovine collagen Source length

kit, which provides native-like 3D collagen gels with the PhotoCol™

917575 Irgacure 2959 UV 365 nm Methanol
-IRG
unique attribute of being tunable when prepared at various
concentrations and crosslinked with blue light. 1X PBS or
PhotoCol™ Blue
916293 LAP 405 nm cell culture
-LAP light
media
The PhotoCol™ kit consists of purified methacrylated Type
Ruthenium 1X PBS or
I bovine collagen as the core component with other support PhotoCol™ Visible 400–
917834 and sodium cell culture
-RUT light 450 nm
reagents in the kit. The methacrylated Type I collagen is persulfate media

produced from telo-peptide intact bovine collagen. The collagen

To sterilize, resuspend and filter each component separately
has been modified by reacting the free amines, primarily the
through a 0.2 micron button filter.
ɛ-amine groups of the lysine residues and the α-amines groups
on the N-termini. Greater than 20% of the total lysine residues Note: The PhotoCol™ kit is designed to provide collagen gels
of the collagen molecule have been methacrylated. The collagen with varying gel stiffness based on collagen concentration and
is extracted from bovine hide and contains a high monomer crosslinking. Light intensity, protein concentration, photoinitiator
content. The collagen starting material was isolated from a closed concentration, photocrosslinking time, and other variables will
herd and purified using controlled manufacturing processes. affect polymerization performance.

The collagen is extracted from bovine hide and contains a high Specifications
monomer content. The collagen starting material was isolated
Storage: Store PhotoCol™ Kits at 2–8 °C. The product ships
from a closed herd and purified using controlled manufacturing
on frozen gel packs. Store the neutralization solution at room
processes.
temperature. Protect from light by storing the bottle in a foil bag
The 20 mM acetic acid solution is provided to solubilize the or wrapping in aluminum foil.
lyophilized methacrylated collagen at concentrations of
Stability: Refer to the expiration date on the batch-specific
3 to 8 mg/ml.
Certificate of Analysis. After solubilizing the collagen with acetic
The neutralization solution consists of an alkaline 10X acid, the collagen solution is stable for 2 months when stored at
phosphate-buffered saline (PBS) solution, which provides 2–8 °C.
physiological salts and neutral pH in the final mixture.
Materials
Photoinitiator solution varies by kit
Materials supplied
Each kit contains a unique photoinitiator that polymerizes under
• Collagen, Type I, methacrylated, lyophilized 100 mg
different conditions and in different solutions.
• Acetic Acid, 20 mM solution 50 ml
• Neutralization solution 10 ml
• Photoinitiator (varies by kit)
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 39

Materials required but not supplied 2. Mix on a shaker table or rotator plate at 2–10 °C until fully
• Cultured cells (visit our website for an up-to-date list of solubilized or overnight. Avoid the formation of air bubbles
cell types) as possible.

• Appropriate cell culture medium Note: The higher concentrations of collagen will take longer to
solubilize.
• PBS (Cat. No. D8537)
• Methanol (if using 917575) 3. Determine the desired volume of collagen required based
on printing requirements.
• Sterile pipette tips for transferring bioink
4. Determine the volume of the neutralization solution (NS) to
• Sterile printing cartridge, piston, and nozzle/needle for 3D
mix with the collagen. To achieve a final pH of 7.0 to 7.4,
printing
follow the guidelines below in Table 2 or Table 3.
• Extrusion-based 3D bioprinter
Note: Dispensing by weight versus volume varies due 1) to the
• Water bath or incubator different viscosity of the different collagen concentrations and 2)
• Micropipettes sample hold up in the pipet tip.

• Light source 5. Transfer the required volume of the neutralization solution

(NS) into a sterile vessel or tube and briefly chill.
Before you start: Important tips for optimal
bioprinting results Note: If the neutralization solution is chilled too long, the salts
Aseptic techniques. Employ aseptic practices to maintain the will come out of solution.
sterility of the product throughout the preparation and handling
6. Calculate the volume of photoinitiator required for
of the collagen and other solutions.
crosslinking following Table 4.
Handling. It is recommended that the collagen and other
Table 2: Collagen to Neutralization Solution by Weight
working solutions be chilled and kept on ice during collagen
preparation.
Solubilized Collagen Weight of Collagen Volume of NS
Vortexing is not recommended unless expressly stated. Concentration

3 mg/ml 1.0 g 100 μl

Procedure 4 mg/ml 1.0 g 114 μl
1. Add volume of 20 mM acetic acid (Table 1) to the 6 mg/ml 1.0 g 120 μl
lyophilized methacrylated collagen to achieve the desired
8 mg/ml 1.0 g 128 μl
concentration.

Table 1: Recommend concentration(s) range from 3 to 8 mg/ml. Table 3: Collagen to Neutralization Solution by Volume

Desired PhotoCol™ Concentration Volume of 20 mM Acetic Acid Solubilized Collagen Volume of Collagen Volume of NS
Concentration
3 mg/ml 33.3 ml
3 mg/ml 1.0 ml 95 μl
4 mg/ml 25.0 ml
4 mg/ml 1.0 ml 90 μl
6 mg/ml 16.7 ml
6 mg/ml 1.0 ml 85 μl
8 mg/ml 12.5 ml
8 mg/ml 1.0 ml 80 μl

Table 4: Photoinitiator Volumes

Cat. No Photoinitiator Calculation for Photoinitiator Volume

916293 LAP Volume of LAP = (Volume of Collagen + Volume of NS) * 0.02

Volume of Ruthenium = (Volume of Collagen + Volume of NS) * 0.02

Volume of Sodium persulfate = (Volume of Collagen + Volume of NS) * 0.02
Ruthenium and sodium
917834
persulfate
Note: Calculate the volume of each photoinitiator. For example, If the resulting number is 100 μl,
you will add 100 μl of ruthenium and 100 μl of sodium persulfate

Calculate Volume of I2959

Volume of I2959 = (Volume of Collagen + Volume of NS) * 0.01
916575 I2959
Note: I2959 only has a 2-week shelf life upon solubilizing. If you need the I2959 to last longer,
remove the required amount and solubilize a 10% solution
40 PhotoCol™ Methacrylated CollagenBioink Preparation

7. Solubilize the photoinitiator with the recommended solvent 11. Load the mixture into a cartridge and dispense or print
in Table 5. the collagen mixture in the desired sterile plates, culture
8. Add the calculated volume of chilled photoinitiator (Table vessels, or molds.
4) to the volume of chilled neutralization solution (NS) 12. Incubate at 37 °C for > 30 minutes for gel formation.
(From Tables 2–3) and mix thoroughly. 13. To photocrosslink, place the gels directly under the
9. Transfer the total volume of the chilled collagen into the recommended light source listed in Table 6.
chilled neutralization solution (NS)/photoinitiator. Mix Note: The consistency and fidelity of photocrosslinking are
quickly and thoroughly by pipetting or rotating a vessel or improved by printing gels on glass-bottom substrates with good
tube. Do not vortex. optical properties that produce minimal light scattering.
Note: Keep the collagen mixture chilled throughout this process.
References
Note: Check to ensure the pH is neutral. The high viscosity of (1) Gaudet, I. D.; Shreiber, D. I. Biointerphases 2012, 7 (1–4), 25.
(2) Drzewiecki, K. E.; Parmar, A. S.; Gaudet, I. D.; Branch, J. R.; Pike, D.
this material can make it difficult to mix.
H.; Nanda, V.; Shreiber, D. I. Langmuir 2014, 30 (37), 11204–11211.

10. If desired, add dispersed chilled cells to the collagen

mixture. Mix quickly and thoroughly by pipetting or
rotating a vessel or tube.
Note: If air bubbles are a concern, allow to sit on ice until the
bubbles come to the surface.

Table 5: Photoinitiator Solutions

Cat. No. Photoinitiator Photoinitiator Volume Final Concentration Solvent

916293 LAP From Step 6 17 mg/ml 1X PBS or cell culture media

Ruthenium and sodium From Step 6 1) Ruthenium: 37.4 mg/ml 1X PBS or cell culture media
917834 2) Sodium persulfate: 119 mg/ml
persulfate

From Step 6 100 mg/ml (Add 1 mL of neat methanol to 100% Methanol

916575 I2959 the amber vial containing 100 mg of I2959
and vortex)

Table 6: Recommended Light Source

Cat. No. Photoinitiator Light Source Wavelength Exposure time

916293 LAP Blue light 405 nm

917834 Ruthenium and sodium persulfate Visible light 400–450 nm

Exposure correlates to gel stiffness*

40 sec 22% increase in stiffness

916575 I2959 UV 365 nm
90 sec 53% increase in stiffness

10 min 75% increase in stiffness

*L
 onger exposure results in more crosslinking; however, exposure to UV and free radicals (generated by the photoinitiator) can affect cellular behavior and lead to cell
death. The effects of exposure length depend on cell type.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 41

PhotoHA™ Methacrylated Hyaluronic Acid

Bioink Preparation
Introduction
Hyaluronic acid is the most abundant glycosaminoglycan in the MilliporeSigma, in partnership with Advanced BioMatrix, offers
body and an essential component of several tissues. While it is PhotoHA™, a purified hyaluronic acid (HA) methacrylate kit,
abundant in extracellular matrices, hyaluronan also contributes which provides native-like 3D HA gels with the unique attributes
to tissue hydrodynamics, movement, and proliferation of cells to be prepared at various concentrations and photocrosslinked
and participates in several cell surface receptor interactions. to provide various gel stiffness.

Hyaluronic acid is a polymer of disaccharides composed of Hyaluronic acid contains primary amino groups that react
d-glucuronic acid and N-acetyl-d-glucosamine, linked via with methacrylic anhydride (MA) to add methacrylate pendant
alternating β-(1→4) and β-(1→3) glycosidic bonds. Hyaluronic groups to the hyaluronic acid molecule. The method renders
acid can be 25,000 disaccharide repeats in length. Hyaluronic the hyaluronic acid into a product with unique properties. The
acid polymers can range from 5,000 to 20,000,000 Da in vivo. PhotoHA™ UV-kit consists of HA methacrylate and a light-
Hyaluronic acid is energetically stable, in part because of the activated photoinitiator.
stereochemistry of its component disaccharides. Bulky groups on
each sugar molecule are in sterically favored positions, whereas The photoinitiator solution will vary by kit
the smaller hydrogens assume the less-favorable axial positions. Each kit contains a unique photoinitiator that polymerizes under
different conditions and in different solutions.

Product Cat. No. Photoinitiator Light Source Wavelength Solution

PhotoHA™-IRG 917079 Irgacure 2959 UV 365 nm Methanol

PhotoHA™-LAP 916471 LAP Blue light 405 nm 1x PBS or cell culture media

PhotoHA™-RUT 917338 Ruthenium and sodium persulfate Visible light 400-450 nm 1X PBS or cell culture media

Specifications
Storage: Store PhotoHA™ Kits at 2–8 °C. The product ships on • PBS (Cat. No. D8537)
frozen gel packs. The product and components are stable for a • Methanol (if using 917079)
minimum of 1 year at receipt in powder form.
• Sterile pipette tips for transferring bioink
Stability: Refer to the expiration date on the batch-specific • Sterile printing cartridge, piston, and nozzle/needle for 3D
Certificate of Analysis. printing
Once solubilized, the PhotoHA™ can be stored at 2-10 °C for • Extrusion-based 3D bioprinter
1 month. The photoinitiator can be stored for no more than 2 • Water bath or incubator
weeks once solubilized
• Micropipettes
Materials • Light source

Materials supplied Before you start: Important tips for optimal

• Methacrylated Hyaluronic Acid, 100 mg bioprinting results
• Photoinitiator (varies by kit) Aseptic techniques. Employ aseptic practices to maintain the
sterility of the product throughout the preparation and handling
Materials required but not supplied of the collagen and other solutions.
• Cultured cells (visit our website for an up-to-date list of
Handling. It is recommended that the collagen and other
cell types)
working solutions be chilled and kept on ice during collagen
• Appropriate cell culture medium preparation.
42 PhotoHA™ Methacrylated Hyaluronic Acid Bioink Preparation

Procedure 4. Solubilize the photoinitiator following Table 2.

The following recommended instructions are for a 1% hyaluronic 5. Add the calculated volume of photoinitiator to the
acid (HA) methacrylate solution. Adjustments to this protocol required volume of HA methacrylate solution and mix until
may be required for various concentrations, recommended homogeneous.
concentrations are 0.5–3.0% (5–30 mg/ml). 6. Resuspend your cell pellet with your HA/Photoinitiator
1. Add 10 ml of 1X phosphate buffer saline (PBS), water, solution
or cell culture media to the 100 mg of lyophilized 7. Dispense or print your HA methacrylate /photoinitiator/
methacrylated HA powder. cell solution into the desired cultureware (i.e., 6-well plate,
2. Mix on a shaker table or rotator plate until fully solubilized 48-well plate), molds, or constructs.
(~30 to 60 minutes) at 2–10 °C. 8. To photocrosslink, place the gels directly under the
Note: Solubilization times may vary depending on the desired recommended light source listed in Table 3.
concentration and volume of PBS, water or medium added. Note: The consistency and fidelity of crosslinking are improved
by printing gels on glass-bottom substrates with good optical
3. Calculate the volume of the photoinitiator required following
properties that produce minimal light scattering.
Table 1.

Table 1: Photoinitiator Volumes

Cat. No. Photoinitiator Calculation for Photoinitiator Volume

916471 LAP Volume of LAP = (Volume of HA solution) * 0.02

Volume of Ruthenium = (Volume of HA solution) * 0.02

Volume of Sodium persulfate = (Volume of HA solution) * 0.02
Ruthenium and sodium
917338
persulfate
Note: Calculate the volume of each photoinitiator. For example, if the resulting number is 100 ul, you will
add 100 ul of ruthenium and 100 ul of sodium persulfate in each vial

Volume of I2959 = (Volume HA solution) * 0.01

917079 I2959
Note: I2959 only has a 2-week shelf-life upon solubilizing. Only dissolve the required amount of
photoinitiator. Store remaining photoinitiator (powder or solution) at 2–10 °C

Table 2. Photoinitiator solutions

Cat. No. Photoinitiator Photoinitiator Volume Final Concentration Solvent

916471 LAP From Step 3 17 mg/ml 1X PBS or cell culture media

Ruthenium and sodium Ruthenium: 37.4 mg/ml

917338 From Step 3 1X PBS or cell culture media
persulfate Sodium persulfate: 119 mg/ml

100 mg/ml (Add 1 mL of neat methanol to the

917079 I2959 From Step 3 amber vial containing 100 mg of I2959 and 100% Methanol
vortex

Table 3: Recommended Light Source

Cat. No. Photoinitiator Light Source Wavelength

916471 LAP Blue light 405nm

Ruthenium and sodium

917338 Visible light 400-450 nm
persulfate

917079 I2959 UV 365nm

*L
 onger exposure results in more crosslinking; however, exposure to UV and free
radicals (generated by the photoinitiator) can affect cellular behavior and lead
to cell death. The effects of exposure length depend on cell type.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 43

Lifeink Type I Collagen Bioink Preparation

®

Introduction
MilliporeSigma, in partnership with Advanced BioMatrix, offers • PBS (Cat. No. D8537)
two types of Lifeinks®. • Sterile pipette tips for transferring bioink

Collagen Bioink Cat. No. • Sterile printing cartridge, piston, and nozzle/needle for 3D
Lifeink® 200, neutralized type I collagen bioink, 35 mg/ml 916226
printing

Lifeink® 240, acidic type I collagen bioink, 35 mg/ml 915211 • Extrusion-based 3D bioprinter
• Water bath or incubator
• Lifeink 200, a bioink that is a highly concentrated
®
• Micropipettes
type I collagen. Lifeink® 200 is pH-neutral collagen with
physiological salt concentration. Lifeink® 200 needs to be Before you start: Important tips for optimal results
printed at 2–8 °C into LifeSupport™ (FRESH Printing) for Aseptic techniques. Employ aseptic practices to maintain the
optimal results. For use in cellular bioprinting. sterility of the product throughout the preparation and handling
• Lifeink® 240 is a Type I collagen bioink at a 35 mg/ml of the collagen and other solutions.
concentration for extrusion-based 3D bioprinting. Lifeink® Handling. It is recommended that the collagen and other
240 is acidified collagen formulated in an acidic saline buffer working solutions be chilled during the preparation of the bioink.
solution. Lifeink® 240 needs to be printed at 2–8 °C into
LifeSupport™ (FRESH Printing) for optimal results. After Vortexing of collagen is not recommended at any step.
printing into LifeSupport™, the pH and salt concentration
Reduce bubble formation. Ensure that NO bubbles enter the
of the printed structure is at physiological levels. Other
system. The introduction of bubbles within the bioink during
materials can be added to the Lifeink® 240 bioink in lieu of
mixing will result in a foam-like material.
cells. Cells should NOT be added directly to this bioink.
Optimize printing conditions. For pneumatic printers, transfer
Specifications the collagen into an appropriate syringe using a syringe coupler.
Storage: Store Lifeink® at 2–8 °C. The product ships on frozen The new syringe should have the seal inserted, but the plunger
gel packs. removed. Centrifuge the syringe at 2000 RPM for 1 minute after
transferring the collagen to remove any air bubbles.
Stability: Refer to the expiration date on the batch-specific
Certificate of Analysis. The product is stable for a minimum of 1
Procedure – Preparing Bioink
year at receipt in powder form.
1. Prepare cell suspension or additives solution (Table 1) for
Materials in a separate sterile syringe (Syringe 1) to be mixed with
the bioink before printing.
Materials supplied
2. Place sterile coupler on the end of Syringe 1 (containing
• Lifeink® 200 or Lifeink® 240
cell suspension or additive solution)
Materials required but not supplied 3. Slowly push plunger until solution forms a slight external
• Cultured cells (visit our website for an up-to-date list of meniscus above the end of the coupler on the syringe.
cell types) (for use with product 916226 only)
4. Remove cap from the syringe with collagen (Syringe 2) and
• Appropriate cell culture medium (for use with product slowly push plunger until collagen forms a slight external
916226 only) meniscus above the end of the syringe.

Table 1: Suspension or additives preparation

Recommended final
Cat. No. Lifeink® Applications Volume of cell suspension or additive solution
concentration

916226 Lifeink® 200 Cell encapsulation 5x106 cells/mL** 2 mL of cell suspension per 5 mL Lifeink® 200*

Additives compatible with 5 mL of additives per

915211 Lifeink® 240 N/A
acidic pH conditions 5 mL LifeInk® 240*
* For smaller volumes, use a similar ratio.
**Cell culture media is recommended for cell suspension.
44 Lifesupport™ Support Slurry Preparation

5. Couple the Syringe 1 (containing the cell suspension or General Printing Notes
additive solution) to the Syringe 2 (collagen). To use a smaller volume of collagen, transfer the desired
Note: Ensure that there are no air bubbles in the system. The amount of collagen to another syringe using the provided sterile
“external meniscus” on both syringes helps prevent air bubbles coupler. To remove the air from the new syringe, you can do
formation. either of the following:

6. Slowly push plungers back and forth ~40 times to ensure • Centrifuge the syringe (capped) with the cap pointing up to
thorough mixing. End with all of the material in the syringe cause the air to accumulate at the cap. Evacuate the air.
to be used for printing. • Centrifuge the syringe (capped) with the cap pointing down,
7. The bioink with other components is now ready for and then use a hemostat to squeeze the syringe while
extrusion 3D bioprinters. pushing the plunger to allow the air to escape.
When printing with FRESH gelatin slurry, allow the final printed
structure to incubate at 37 °C for 30 to 60 minutes, and then
replace the gelatin with media.

Lifesupport™ Support Slurry Preparation

Introduction Specifications
FRESH 3D bioprinting is performed by extruding bioinks and Storage: Store LifeSupport™ at room temperature. Limit
other materials within the hydrated, compacted LifeSupport™ exposure to air, LifeSupport™ is highly hygroscopic.
bath, specially formulated to prevent constructs from
Stability: Refer to the expiration date on the batch-specific
collapsing and deforming during printing. A wide range of
Certificate of Analysis. Rehydrated LifeSupport™ can be stored in
polymer crosslinking chemistries and gelation mechanisms
the noncompacted state (i.e., before centrifugation) for 7 days
can be supported within LifeSupport™ by incorporating ions,
under refrigeration to avoid degradation.
enzymes, pH buffers, and more into the support bath during
the rehydration process. LifeSupport™ allows for FRESH 3D Once compacted, LifeSupport™ should be used within 12 hours,
bioprinting of soft hydrogel bioinks in complex geometries and the temperature should not exceed 32 °C during handling or
without the need for sacrificial support inks (e.g., Pluronic® printing.
F-127, polycaprolactone, gelatin) or ink modifiers to increase
mechanical stability (e.g., gelatin methacrylate, cellulose, Materials
alginate).
Materials supplied
LifeSupport™ can be rehydrated in a range of buffers and cell Each LifeSupport™ printing kit comes with 5 individual 2 g units
culture media to support multiple cell types and specific bioinks. of sterile, dried, LifeSupport™ powder, composed of gelatin
LifeSupport™ can also be rehydrated to support the cross- microparticles of defined size and shape. Each unit rehydrates to
linking and/or gelation of multiple types of bioinks within the approximately 20 mL of support bath.
same container of support bath. Bioinks that can be printed into
the support bath include collagen, alginate, fibrin, decellularized Materials required but not supplied
extracellular matrix, methacrylated gelatin, methacrylated • Cultured cells (visit our website for an up-to-date list of
hyaluronic acid, and more. The specific bioinks that can be cell types) (for use with product 916226 only)
printed will also depend on the hardware capabilities of the 3D
• Appropriate cell culture medium (for use with product
bioprinter that you are using.
916226 only)
MilliporeSigma, in partnership with Advanced BioMatrix, • PBS (Cat. No. D8537)
offers two types of Lifeinks® that are formulated to print into
• Sterile pipette tips for transferring bioink
LifeSupport™ as discussed in the previous protocol.
• Sterile printing cartridge, piston, and nozzle/needle for 3D
Description Cat. No. printing
LifeSupport™, support slurry for FRESH bioprinting 915467 • Extrusion-based 3D bioprinter
• Water bath or incubator
• Micropipettes
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 45

Before you start: Important tips for optimal 6. Dislodge the compacted LifeSupport™ slurry by holding
bioprinting results horizontally, and gently tapping the body of the tube
Aseptic techniques. Employ aseptic practices to maintain the against the edge of a hard surface 15 times (Figure D).
sterility of the product throughout the preparation and handling 7. Shake the tube containing dislodged LifeSupport™
of the collagen and other solutions. vigorously for 10 seconds. Shake along the length of the
tube (Figure D). User should feel LifeSupport™ moving
Handling. To prevent premature melting of LifeSupport™, all
and hitting the cap and inner surfaces of the tube during
suspension media should be refrigerated before use.
shaking.
Reduce bubble formation. Ensure that NO bubbles enter the Note: Hold tube by the cap during shaking and avoid handling
system. The introduction of bubbles within the bioink during the body of the tube to reduce warming.
mixing will result in a foam-like material.

Optimize printing conditions. For pneumatic printers, transfer

the collagen into an appropriate syringe using the coupler. The
new syringe should have the seal inserted, but the plunger
removed. Centrifuge the syringe at 2000 RPM for 1 minute after
transferring the collagen to remove any air bubbles.

Procedure
1. For best preparation results, we strongly recommend
splitting the 2g unit of LifeSupport™ into 1g aliquots. Add
35 mL of cold (4 °C) suspension media to a 1g aliquot of
LifeSupport™.
2. Vortex (Figure A) and shake vigorously for 1 min to ensure
Figure B. LifeSupport™ compacted at the bottom of the centrifuge tube
all powder is fully resuspended (top right) and not stuck to
with liquid supernatant on top.
tube walls / tip (bottom right).
3. Let stand for 10 minutes at 4 °C to allow LifeSupport™ to
rehydrate fully.
OPTIONAL: Degas the support bath in a vacuum chamber for
30 min to remove dissolved gases and prevent the formation of
bubbles during printing.

4. After rehydration, shake LifeSupport™ for 10 seconds.

Centrifuge the rehydrated LifeSupport™ at 600x g for
5 min.
5. The LifeSupport™ should now be compacted at the
bottom of the centrifuge tube (Figure B). Gently pour
Figure C. LifeSupport™ compacted at the bottom of the centrifuge tube
off or aspirate the liquid supernatant to leave only the with liquid supernatant removed.
compacted LifeSupport™ in the bottom of the centrifuge
tube (Figure C). Cap the tube containing the compacted
LifeSupport™. TAP

Figure D. Demonstrating the tapping process.

250

3
Figure A. Proper vortexing
46 Lifesupport™ Support Slurry Preparation

8. Centrifuge well-shaken LifeSupport™ at 1000x g for 5 min 12. The bath should be as bubble-free as possible. Tapping
to compact it. If resuspended in serum-based growth firmly against the table can force large bubbles to the
media, centrifuge at 2000 x g for 5 min until compacted. surface. The bath should not move easily if the container
To ensure the LifeSupport™ bath is compacted the material is tilted. Be gentle when tapping glass dishes. It is
should stay in place when the tube is slowly turned on its recommended that you use a print container that provides
side (Figure E). a minimum of 1 mm clearance on the bottom and a
Note: Use a temperature-controlled centrifuge if possible. If minimum of 3 mm clearance on all sides as well as the top
this is not available, carefully monitor LifeSupport™ behavior of the construct to be printed. Additional clearance is fine
after centrifugation. If your centrifuge warms up significantly but requires using more LifeSupport™.
during the centrifugation cycle, it may affect the performance of Printing Recommendations
LifeSupport™.
Note: LifeSupport™ can be used as a scaffold support for a
9. The LifeSupport should now be compacted at the
™ variety of bioinks, including Lifeink® 200, Lifeink® 240, collagen,
bottom of the centrifuge tube. Gently pour off or aspirate alginate, fibrinogen, and other inks with cells. For recommended
any remaining liquid supernatant to leave only the printing guidelines for Lifeink®, please refer to the protocols
compacted LifeSupport™ in the bottom of the centrifuge within this guide.
tube. LifeSupport™ has been appropriately prepared if
1. Ensure the print container is large enough to avoid the
the LifeSupport™ stays in place when the tube is slowly
needle running into the walls during printing.
placed horizontally (Figure E). A small amount of flow is
acceptable. 2. Place the LifeSupport™ bath on your 3D bioprintering
platform. OPTIONAL Vacuum grease (Dow Corning,
WARNING If the LifeSupport™ flows easily in the tube
1597418) can be added to the bottom of the print container
(Figure E), stop and resuspend in cold media, and repeat
to prevent sliding during printing.
steps 4–10. In this case, it may be necessary to increase the
“2nd Centrifugation” speed in step 9 in 200X g increments until 3. Position your needle ~1 mm off the bottom of the
LifeSupport™ is adequately compacted. container, then move the needle to the middle of the
container. Unlike typical printing, the needle does not
10. Aseptically remove the compacted LifeSupport™ using a have to start out touching or even be close to the bottom
sterile spatula into the desired printing container. of the container. The support bath will trap your print in
11. Tap the container against a surface to settle and evenly place no matter where you start. Ensure that your printer
distribute the bath in the container. begins printing from this position. You may need to disable
homing procedures to prevent the printer from traveling
outside the container.
4. Begin printing.

Print Release Recommendations

1. After printing, incubate at 37 °C for at least 30 min to
release your print.
2. After 30 min of incubation, the LifeSupport™ bath should be
fully melted, and your printed structure will be released.
Large volumes may require longer times to completely
Figure E. Demonstrating properly compacted LifeSupport™. melt.
3. Carefully transfer released prints into warm (37 °C)
suspension media according to your ink.
NOTE: Melted LifeSupport™ can be serially replaced with
suspension media to avoid handling the printed construct. For
example, if you printed into a 6-wellplate, carefully aspirate 2
mL of melted LifeSupport™ out and add 2 mL of warm media.
Repeat this process until most of the gelatin has been replaced
by media.

4. If culturing tissues, continue standard media exchange in

Figure F. Removing compacted LifeSupport™. accordance with your cell culture protocol.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 47

Decellularized ECM Bioink Precursor

Preparation
Introduction Before you start: Important tips for optimal
MilliporeSigma, in partnership with T&R Biofab, offers a bioprinting results
decellularized ECM bioink precursor. The decellularized ECM Aseptic techniques. Employ aseptic practices to maintain the
(dECM) is a biomaterial consisting of both structural and sterility of the product throughout the preparation and handling
functional biomolecules, such as collagen, glycosaminoglycans, of the collagen and other solutions.
and glycoproteins. Decellularized ECM bioink precursors have
Handling. All processes should be completed at 2–8 °C to
different characteristics depending on the origin of tissues (skin,
prevent gelation of the solubilized bioink precursor.
bone, cartilage), thus providing optimized environments for
cellular activities that are tissue-specific. The solubilized bioink should be prepared and used immediately

Decellularized Bioink Cat. No. Procedure

Decellularized ECM bioink precursor from porcine bone,
suitable for 3D bioprinting applications
906883 Bioink Solubilization process
Decellularized ECM bioink precursor from porcine skin, 1. Prepare 0.5M acetic acid and decellularized ECM bioink
906867
suitable for 3D bioprinting applications precursor on ice.
Decellularized ECM bioink precursor, from porcine
cartilage, suitable for 3D bioprinting applications
906875 2. Place the decellularized ECM bioink precursor in a glass vial
and add 0.5M acetic acid to the desired concentration. A
3% ink (30 mg/ml) is recommended for printing. (i.e. 1 ml
Specifications final sample; 24 mg precursor in 800µl of acetic acid)
Storage: Store decellularized ECM bioink precursor at -20 °C for
3. Keep the sample at 2–8 °C for 72 hours to ensure
up to 12 months.
solubilization. (Figure 1 and 2)
Stability: Refer to the expiration date on the batch-specific Note: It is recommended to Vortex ink every 24 hours during
Certificate of Analysis. the solubilization process.

Materials
Materials supplied 0.5M
Acetic acid ink
• Decellularized ECM bioink precursor lyophilized solid 100 mg

Materials required but not supplied Bioink

precursor at 4° C
• 0.5M acetic acid for 72hr

• 10× Minimum Essential Medium (MEM) (Cat. No. M0275)

• HEPES (Cat. No. H4034)
• Sodium bicarbonate (NaHCO3)
• Deionized or Distilled water Figure 1. Bioink Solubilization process

• Sodium hydroxide (NaOH)

• Cultured cells (visit our website for an up-to-date list of 10x MEM RB buffer
cell types)
• Appropriate cell culture medium 250
Recommendation NaHCo3
• PBS Sigma-Aldrich HEPES
(Cat. No. M0275) Distilled water
• Sterile tube for reagent preparation NaOH

• Positive displacement pipette is recommended

• Sterile pipette tips for transferring bioink
It is Recommended to use RB buffer
• Sterile printing cartridge, piston, and nozzle/needle for 3D within 7 days after preparation
printing
Figure 2. Reagent preparation
• Extrusion-based 3D bioprinter
• Petri-dish
• Scraper
• pH indicator
250
48 Decellularized ECM Bioink Precursor Preparation

Reagent preparation 5. Place RB, 10xMEM (Minimum Essential Medium, Cat. No.
4. Prepare resuspension buffer (RB) by mixing the comments M0275) and solubilized bioink precursor on ice to cool for
as mentioned in Table 1. ~10 mins
6. Mix together 10 MEM, RB, and ink (mixing volume ratio -
Bioink preparation procedure
ink:10MEM:RB = 8:1:1) (i.e. 800 µl ink, 100 µl 10X MEM,
Note: This procedure can be done using either a petri dish or a
100 µl RB).
50 mL tube (Options 1 and 2).
7. Adjust ink pH to 6.5-7.0. See Options for mixing in
Table 1. RB composition following sections.
Note: The color of the final bioink should be light orange
NaHCo3 HEPES Distilled NaOH (Figure 1). If the pH of the bioink is too low (pH <6.5, and
(g) (g) water (ml) (g) indicated by light yellow or yellow color), add small quantities
Skin 906867 0.22 0.48 10    1.4 (1-2 µl) of RB reagent and gradually adjust it to the ideal
Bone 906883 0.22 0.48 10 1.7 pH range.
Cartilage 906875 0.22 0.48 10 1.7
8. Resuspend the cell pellet at the desired cell density with
Note: Use RB buffer within 7 days after preparation. the bioink solution by gently pipetting up and down.
Maximum 100 µl cell suspension can be mixed with 1 ml
Option 1: Prepare in a petri dish
of bioink solution; we recommend a starting cell density of
1x107 cells/ml media, although this will vary depending on
RB buffer 10X MEM Mix RB buffer the cell type used.

Printing procedure
Positive displacement
pipette
9. Transfer solubilized bioink precursor to a printing cartridge
Solubilized
bioink
precursor (Figure 2).

Petri dish
Note: Centrifugation can be used for removing residual bubbles
pH is too low (light yellow)
(Recommended condition: 2,000 RPM for 30 s at 4 °C)
Mixing volume ratio gradually add small
Ink: 10xMEM: RB = 8: 1: 1 quantities (1-2 µL) of
RB reagent 10. Start printing process using bioink, and then incubate the
printed construct for 40-60 minutes without any solutions
Mix Mix
(ex. culture medium) in an incubator (at 37 °C in a
Cell
humidified atmosphere with 5% CO2) for gelation.
Note: Recommending printing temperature: For prints < 30
minutes: room temperature printing, for prints >30mins <3
hours: 15 °C.

Option 2: Prepare in a 50mL tube

Figure 1. Mixing to correct pH adjusted color.
250
RB buffer 10X MEM
Positive
displacement

360
pipette 10X MEM
pH-adjusted ink
RB

Vertexing centrifuge Vertexing centrifuge

Ink

450

50ml tube Figure 2. Printing Procedure

RB buffer
Cell Positive 250
displacement pipette

Vertexing centrifuge Vertexing centrifuge

Centrifuge

Printing
pH is too low (light yellow) pH 6.5-7
gradually add small
Printing
quantities (1-2 µL) of
syringe
RB reagent

Syringe cap Printing nozzle

250
250
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 49

Example of printing conditions; printing conditions may vary Example

from user to user.
Printer: Cellink BIO X™ or Cellink INKREDIBLE™ printer
Contents Skin Bone Cartilage

Ink concentration Temperature: 15 °C

3%
(w/v %)

Nozzle Diameter
Flow rate (speed): 10 mm/s
500
(µm)
Nozzle: 22G TT tapered needle
Minimum pneumatic
10
pressure (kPa)
Pressure: 50–60 kPa for Cartilage and Bone bioinks
Feed rate
500
(mm/min)
Culture cells
Strut width
700 730 950 After the printed construct has been incubated, add the
(µm)
appropriate cell culture medium and culture the bioprinted tissue
with the appropriate cell culture medium following standard
Representative tissue culture procedures.
images

INVIVO-Gel Preparation
Introduction Materials
The INVIVO GEL product is made from gelatin methacrylate Materials supplied
(GelMA) created to mimic the extracellular environment. INVIVO
• INVIVO-Gel (5 ml x 2 ea)
GEL bioink series has two distinct advantages compared to
other commercially available GELMA based bioink products. • mixing tube (1 ea)
First, INVIVO GEL provides a degree of freedom to tune the • 0.22 um filter (1 ea)
elastic modulus (or stiffness) according to user’s specific needs • photo-initiator agent (powder form)
by changing the concentration of INVIVO GEL (GELMA) and Gel-
linker, a photo-initiator. Also, by changing the UV light (365 nm) Materials required, but not supplied
exposure time, the degree of cross-linking can be adjusted. The • Cultured cells (visit our website for an up-to-date list of
INVIVO GEL formulation mimics extracellular environment with cell types)
a choice of growth factors (VEGF A, TGF-β1, BMP-2, or GF),
• Appropriate cell culture medium
allowing precise control of the extracellular environment in your
• PBS (Cat. No. D8537)
research.
• Sterile pipette tips for transferring bioink
MilliporeSigma in partnership with ROKIT now offers newly and
• Sterile printing cartridge, piston, and nozzle/needle for 3D
uniquely formulated GELMA based bioinks.
printing
Name Cat. No.
• Extrusion-based 3D bioprinter
INVIVO GEL BMP-2 bioink 923745
• Water bath or incubator
INVIVO GEL essential bioink 923796
• Micropipettes
INVIVO GEL TGF-ß bioink 923753

INVIVO GEL VEGF bioink 923737

Before you start: Important tips for optimal results
Aseptic techniques. Employ aseptic practices to maintain the
Specifications
sterility of the product throughout the preparation and handling
Storage: Store INVIVO GEL at 2–8 °C. The product ships on of the collagen and other solutions.
frozen gel packs.
Handling. It is recommended that working solutions be chilled
Stability: Refer to the expiration date on the batch-specific during the preparation of the collagen.
Certificate of Analysis. The product is stable for a minimum of 1
year at receipt in powder form.
50 INVIVO-Gel Preparation

Procedure – Photoinitiator Solution Cell Preparation

Preparation (in a dark room). 11. Prepare a cell pellet and remove the supernatant as much
1. Add 500 ul of room temperature sterile distilled water to as possible.
the photo-initiator vial. 12. Recommended cell density is 1 x 10 6 cells/ml
2. Close the vial cap and mix well to dissolve. 13. Add 1 ml of the INVIVO-Gel solution.
3. Sterilize the photoinitiator solution by filtering through the 14. Resuspend the cell pellet with INVIVO-Gel. (If possible, cut
0.22 um filter (included). the tip of a sterile 1000 µl micropipette tip while keeping
4. Aliquot the photoinitiator solution into covered or opaque sterility before cell resuspension to reduce stress on the
tubes and store at -20 °C or -80 °C until use. cells.)

5. Use 35 µl of photo-initiator solution per 1 ml of INVIVO- 15. Mix together the 1 ml cell mixed INVIVO-GEL back to
Gel. For 5 ml INVIVO-Gel, 175 µl of photoinitiator is the 4ml INVIVO-Gel in the syringe (a mixing tube is
required. recommended).
16. Incubate the final cell-laden INVIVO-Gel at 4 °C for 10
Warming INVIVO-Gel
minutes before starting the printing process.
6. Warm the INVIVO GEL in a 37 °C water bath for about 10
17. Install the INVIVO-GEL to the printer with the following
min until it turns into a clear yellowish color liquid
setting and rest for 10 minutes before start of printing.
Combining INVIVO-Gel with the Photoinitiator
Printing
Agent (in a dark room)
7. After warming INVIVO-GEL, spray 70% ethanol to the Recommended ROKIT Dr. 4D2 Bioprinter
INVIVO-Gel syringe and wipe before proceeding.
parameter setting.
Note: Parameters with other printers may need to be optimized.
8. Remove the cap from the INVIVO-Gel syringe and connect
a mixing tube. • Syringe holder: 10 °C
9. Take 175 µl of the sterilized photoinitiator solution. While • Bed: 8 °C
carefully pulling the piston of the INVIVO-Gel syringe
• Average printing speed: 3 mm/s
down and removing air inside the mixing tube, add the
photoinitiator solution into the tube by careful pipetting. • Max printing speed: 5 mm/s

10. Connect an empty syringe to the other side of the mixing • Nozzle size: 0.2 mm
tube. Mix carefully by moving back and forth between the • Height per layer: 0.15 mm
syringes. • Printing feature diameter: 10–15 mm
Note: After adding photo initiator, pull the plunger until the
photoinitiator-added INVIVO-Gel solution comes to the edge of
the mixing tube so you can minimize air bubble formation during
mixing.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 51

Cell-bioink Mixing Protocol

with TissueFab Bioinks
®

Introduction Protocol A: For Pipetting or Low Viscosity

Bioinks are generally provided in an acellular form, but Bioinks
researchers may be interested in incorporating their specific
Prepare bioink
viable cells of interest to create a cell-laden printed construct.
Warm the 10 mL bottle of TissueFab® bioink in a water bath
This is usually carried out in one of two methods outlined
or incubator set to 37 °C for 30 minutes or until the bioink
below, depending on the material properties of the bioink. If
becomes fluid and easy to pipette.
the bioink can be liquified through temperature or other means,
Protocol A will be suitable through pipetting the cell pellet
Prepare bioink-cell solution
with the bioink in its low viscosity state. If the bioink is highly
1. Centrifuge the cell suspension to obtain a cell pellet.
viscous or preloaded into a sterile syringe cartridge, Protocol
Remove the supernatant carefully so that the cell pellet is
B is recommended using a cellmixer. The final cell density will
not disrupted.
depend on the application; however, 1–10 x 10 6 cells/mL bioink is
recommended. 2. Resuspend the cell pellet at the desired cell density with
the bioink solution by gently and slowly pipetting up and
Disclaimer down several times.
TissueFab® bioinks are for research use only, not suitable for a. Ensure the cells are evenly distributed in the bioink
human, animal, or other use. Please consult the Safety Data solution by gently and slowly pipetting up and down
Sheet for information regarding hazards and safe handling several more times. Avoid creating air bubbles. DO NOT
practices. vortex or shake vigorously. Be careful not to dilute the
bioink solution with the cell culture medium because it
Specifications may interfere with the printability of the bioink.
Storage: Store TissueFab® bioinks at 2–8 °C. Protect from light
3. Pipette the bioink-cell solution into the desired printing
by storing the bottle in a foil bag or wrapping in aluminum foil.
cartridge. This step creates a filled printing cartridge.
Stability: Refer to the expiration date on the batch-specific 4. Place the remaining bioink in a foil bag or wrap in
Certificate of Analysis. aluminum foil and store at 4 °C to protect from heat and
light.
Materials
Materials supplied
TissueFab® bioinks are supplied as follows:

• 1 × 10 mL bottle (1 unit)

Materials required but not supplied

• Cultured cells (visit our website for an up-to-date list of
cell types)
• Appropriate cell culture medium
• PBS (Cat. No. D8537)
• Sterile pipette tips for transferring bioink
• Sterile printing cartridge, piston, and nozzle/needle for 3D
printing
• Extrusion-based 3D bioprinter
• Water bath or incubator
• Cell mixing unit (for mixing large amounts of bioinks)
• Female/female Luer lock adaptors
• Micropipettes
• Light source
52 Cell-bioink Mixing Protocolwith TissueFab® Bioinks

Protocol B: For Preloaded Syringe or High To Prepare 3+ ml using a cell mixer

Viscosity Bioinks 1. Prepare the cell suspension – Resuspend 10 million cells
per 100 µL cell culture medium.
Prepare bioink
a. Recommend 100ul of cells per 1 ml of bioink.
Warm the 10 mL bottle of TissueFab® bioink in a water bath
or incubator set to 37 °C for 30 minutes or until the bioink 2. Use a 10:1 bioink:cell suspension, taking care not to
becomes fluid and easy to pipette. introduce air bubbles to the mixture. Transfer bioink and
cell suspension into separate female/female Luer lock
To Prepare 1-2 ml using female/female Luer lock adaptors. Attach syringes to a cell mixing dispensing unit.
adaptors
3. Connect the bioink and cell suspension syringes to a
1. Prepare the cell suspension – Resuspend 10 million cells
mixing unit, then connect the empty cartridge to the other
per 100 µL cell culture medium.
side of the mixing unit.
a. Recommend 100ul of cells per 1 ml of bioink.
4. Apply gentle pressure onto the dispensing unit to mix the
2. Use a 10:1 bioink:cell suspension, taking care not to content of both syringes into the empty cartridge.
introduce air bubbles to the mixture. Transfer bioink and
cell suspension into separate female/female Luer lock
adaptors. Attach the bioink syringe to the syringe with cell
suspension.
3. Carefully mix the bioink with the cell suspension by gently
pushing the bioink back and forth between the syringes.
Transfer the cell containing bioink back to the cartridge
and cap it.
Note: To avoid an air gap when mixing, pre-fill the Luer lock
adaptor with a small amount of bioink of choice before attaching
the syringe with the cell suspension.
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 53

General Guide to Photo, Ionic, and

Enzymatic Crosslinking
Introduction Before you start: Important tips for optimal
Bioinks are mainly composed of hydrogels or soft materials. bioprinting results
In the process of 3D bioprinting, bioinks are constructed into Optimize printing conditions. Optimize printing conditions (e.g.,
3D structures. To keep the integrity of the printed structure, nozzle diameter, printing speed, printing pressure, temperature,
crosslinking is utilized during or post printing. Crosslinking cell density) for the features of your 3D printer and for your
significantly increases the mechanical strength of the application to ensure successful bioprinting. The suggestions
bioprinted constructs, preventing them from collapsing or below can guide you.
dissolving due to environmental changes such as temperatures
Reduce bubble formation. If the bioink has air bubbles, the
or the addition of media. It also affects the physicochemical
bubbles may hamper bioprinting. Carefully handle the bioink
properties of the bioprinted constructs and the cellular
when you mix and transfer to avoid bubble formation. Do not
behavior of encapsulated cells.
vortex or shake vigorously.
Photocrosslinking is widely used in 3D bioprinting for its ease
Aseptic techniques. Follow standard aseptic handling
in preparation and operation. The choice of photoinitiator
techniques when preparing and printing the bioink and during
determines the wavelength of light needed to crosslink the
cell culture.
bioink. During photocrosslinking, the light wavelength and
exposure time can impact cell viability and other cellular Cell density. Resuspend the cell pellet to the appropriate
behaviors. volume for the desired printed structure and cell density. Typical
cell density for extrusion-based bioprinting is 1 to 5 × 10 6
Disclaimer cells/ mL.
TissueFab® bioinks are for research use only, not suitable for
Note:The number of prints obtained from each 10 mL bottle of
human, animal, or other use. Please consult the Safety Data
bioink (a unit) varies depending on the printed structure. For
Sheet for information regarding hazards and safe handling
example, each 10 mL bottle contains enough material to print a
practices.
30 µL structure in each well of three 96-well plates or a 100 µL
Specifications structure in each well of four 24-well plates.

Storage:Store TissueFab® bioinks at 2–8 °C. Protect from light

Procedure
by storing the bottle in a foil bag or wrapping in aluminum foil.
UV Light Crosslinking
Stability:Refer to the expiration date on the batch-specific
1. Calibrate the irradiance of light if needed.
Certificate of Analysis.
2. Place the UV light source directly above the 3D-bioprinted
Materials structure and expose the structure to the UV light.

Materials supplied 3. Use the appropriate distance and exposure time based on
your light source. For low-intensity light sources commonly
TissueFab® bioinks are supplied as follows:
available in desktop bioprinters, such as Cellink™
• 1 × 10 mL bottle (1 unit) bioprinters (Bio X™ and INKREDIBLE™ printers), distances
of 3 cm or less and exposure times of 60 s or more are
Materials required but not supplied
recommended.
• Cultured cells (visit our website for an up-to-date list of
Note: Recommended settings for bioinks:
cell types)
• Appropriate cell culture medium wavelength – 365 nm; irradiance – 10 mW/cm2; exposure – 90s

• PBS (Cat. No. D8537) 4. The 3D-bioprinted structure is ready for culture or analysis
• Sterile pipette tips for transferring bioink immediately after crosslinking.

• Sterile printing cartridge, piston, and nozzle/needle for 3D

printing
• Extrusion-based 3D bioprinter
• Water bath or incubator
• Micropipettes
• Light source
54 General Guide to Photo, Ionic, and Enzymatic Crosslinking

TissueFab® Inks for UV Crosslinking 1. Gently pipette the crosslinking solution on the
Cat. No. Product Name 3D-bioprinted construct. Ensure the entire structure is
covered by the solution. The crosslinking time may vary
905410 TissueFab® bioink Alg(Gel)ma -UV/365 nm
depending on the structure size.
905429 TissueFab® bioink (Gel)ma-UV/365 nm
Note: Recommended settings for crosslinking solution: 200mM
919632 TissueFab bioink
®
(GelHA)ma -UV/365 nm
CaCl2 (available as 919926 TissueFab® crosslinking solution;
920983 TissueFab® bioink (GelAlg)ma -UV/365 nm crosslinking time: 1 min).
920975 TissueFab bioink
®
(GelAlgHA)ma -UV/365 nm
2. Remove the crosslinking solution by washing twice with
915025 TissueFab bioink Bone
®
UV/365 nm
PBS.
915726 TissueFab® bioink Conductive UV/365 nm
3. Add cell culture media and incubate.
4. The 3D-bioprinted structure is ready for culture or analysis
Visible light crosslinking
immediately after crosslinking.
1. Calibrate the irradiance of light if needed.
Cat. No. Product Name
2. Place the light source directly above the 3D-bioprinted
structure and expose the structure to the specific 906913 TissueFab bioink
®
Alg(Gel) ma -Vis/525 nm
wavelength of light. 905410 TissueFab bioink
®
Alg(Gel) ma -UV/365 nm
Note: Use the appropriate distance and exposure time based on
your light source. For 405 nm light sources commonly available Enzymatic crosslinking -Thrombin crosslinking
in desktop bioprinters, such as Cellink™ bioprinters (Bio X™ and
Enzymes such as microbial transglutaminase, tyrosinase, and
INKREDIBLE™ printers), distances of 3 cm or less and exposure
thrombin are commonly used as catalysts for crosslinking in 3D
times of 30–40 s or more are recommended.  
bioprinting due to their biocompatibility. Thrombin plays a vital
Note: Recommended settings for inks: role in regulating the conversion of fibrinogen to fibrin monomer
and further polymerizing to form insoluble fibrin clots. It is used
wavelength – 405 nm; irradiance – 10 mW/cm2; exposure – 30s. as an enzymatic crosslinker in 3D bioprinting bioinks containing
Wavelength – 525 nm or white light; power – 800 mW/cm2; fibrinogen. Fibrinogen promotes cell adhesion, proliferation
distance – 8 cm; exposure – 60s and migration, and therefore, has been widely utilized in tissue
engineering for wound healing, neural regeneration, bone
3. The 3D-bioprinted structure is ready for culture or analysis generation and vascularization, fibrinogen containing gels, and
immediately after crosslinking. more.
405 nm TissueFab® Inks for UV Crosslinking
1. Prepare 1 unit/uL thrombin stock solution by adding 100 uL
Cat. No. Product Name DPBS to 100 units lyophilized thrombin. The stock solution
919624 TissueFab® bioink (GelHA)ma -Vis/405 nm is recommended to be stored at -80 °C.
918741 TissueFab bioink
®
(Gel) ma -Vis/405nm 2. Prepare 10units/ml thrombin solution in cell culture media
921610 TissueFab® bioink (GelAlg) ma -Vis/405 nm by diluting 1 unit/uL thrombin stock solution 100 times with
cell culture media (e.g., 10 uL thrombin stock solution +
922862 TissueFab® bioink (GelAlgHA) ma -Vis/405 nm
990 uL cell culture media).
915033 TissueFab® bioink Bone Vis/405 nm
4. Gently pipette the diluted thrombin solution on the
915963 TissueFab® bioink Conductive Vis/405 nm
3D-bioprinted construct. Ensure the entire structure is
525 nm TissueFab® Inks for UV Crosslinking
covered by solution.

Cat. No. Product Name 5. Change the media after overnight incubation or until the
next standard media change time.
906913 TissueFab® bioink Alg(Gel)ma -Vis/525 nm
6. The 3D-bioprinted structure is ready for culture or analysis
immediately after crosslinking is complete.
Ionic crosslinking
Ionic crosslinking is one of the most common crosslinking References
methods used in 3D bioprinting, and it is commonly used GhavamiNejad, A.; Ashammakhi, N.; Wu, X. Y.; Khademhosseini, A. Small
2020, 16 (35), 2002931.
for alginate and its derivatives. Alginate is a water-soluble
(Khoon, L. S.; Galarraga, J. H.; Xaiolin, C.; Lindberg, G. C. J.; Burdick, J. A.;
polysaccharide composed of linked units of β-l-guluronate Woodfield, T. B. F. Chem. Rev. 2020, 120 (19), 10662–10694.
(G) and α-d-mannuronate (M). In the presence of multivalent Xiaolin, C.; Li, J.; Hartanto, Y.; Durham, M. Adv. Healthc. Mater. 2020, 9 (15),
1901648.
cations (such as Ca2+), carboxylic groups of adjacent alginate
Teixeira, L.S. M.; Feijen, J.; van Blitterswijk, C. A.; Dijkstra, P. J.; Katerien, K.
polymer chains are bonded in exchange of sodium ions to form Biomaterials 2012, 33 (5), 1281–1290.
crosslinked networks.
Polymers with
Possibilities
Functionalized Poly(ethylene glycol)s
for Drug Delivery
Poly(ethylene glycol)
Polymer of choice for optimal and
reproducible results.
When it comes to drug delivery
O H
technologies and solutions, poly(ethylene H O
glycol)s or PEGs are the polymer of choice
for optimal and reproducible results. With n
excellent pharmacokinetic properties,
they are ideal materials for bioconjugation, peglylation, crosslinking, and
hydrogel formation.
Let us help you transform your work into new therapeutic discoveries with
our diverse PEG selection.

Features
• Well characterized, high-purity materials with a wide variety of functional groups
• High biocompatibility, with little to no immunogenicity
• Mn ranging from 1-40 kDa
• Reactivity
- For amine, N-terminal amine, and thiol pegylation
- For click chemistry and photochemistry
- Heterobifunctional and multi-arm PEG crosslinkers
• Narrow polydispersity

For a complete list of available

materials, visit: SigmaAldrich.com/PEG

The life science

business of Merck
operates as
MilliporeSigma in
the U.S. and Canada.
56 Natural Polymers

Natural Polymers
Cellulose Precursors
Name Structure Molecular Weight Extent Of Labeling Cat. No.
2-Hydroxyethyl cellulose RO Average Mv ~90,000 2.5 mol per 1 mol (M.S.) 434965-250G
434965-1KG
O
RO OR Average Mw ~380,000 2.0 mol per 1 mol (M.S.) 308633-25G
R = H or 1.0 mol per 1 mol (D.S.) 308633-500G
OO O R
OR OR * H Average Mv ~720,000 2.5 mol per 1 mol (M.S.) 434973-250G
O
x
434973-1KG
RO OR n Average Mv ~1,300,000 2.5 mol per 1 mol (M.S.) 434981-250G
434981-1KG
Hydroxyethylcellulose RO - - 525944-50G
ethoxylate, quaternized R = H or
O * H or
RO OR O
x
OO O R
Cl
CH3
OR H
OR * N
O
y CH3
RO OR OH
n

Hydroxypropyl cellulose RO Average Mn ~10,000 - 435007-5G

R = H or Average Mw ~80,000 435007-100G
OO R 435007-250G
RO OR CH3
Average Mw ~100,000 - 191884-5G
OO *
O H 191884-100G
OR OR x 191884-250G
RO OR Average Mw ~370,000 - 191892-5G
n
191892-100G
191892-250G
Average Mw ~1,000,000 - 191906-5G
191906-100G
191906-250G
(Hydroxypropyl)methyl RO Average Mn ~10,000 Methoxy 1.8–2.0 mol per 1 423238-25G
cellulose mol (D.S.) 423238-100G
O R = H or CH3 or Propylene oxide 0.2–0.3 mol
RO OR per 1 mol (M.S.)
CH3 Methoxy 29 wt. %
OO O R
OR OR * Propylene oxide 7 wt. %
O H
RO x Average Mn ~86,000 Methoxy 1.8–2.0 mol per 1 423203-100G
OR n mol (D.S.)
Propylene oxide 0.2 mol per
1 mol (M.S.)
methoxy 29 wt. %
propylene oxide 7 wt. %
Average Mn ~90,000 Methoxy 1.1–1.6 mol per 1 423181-100G
mol (D.S.)
Propylene oxide 0.1–0.3 mol
per 1 mol (M.S.)
Methoxy 21 wt. %
Ppropylene oxide 5 wt. %
Average Mn ~120,000 Methoxy 1.1–1.6 mol per 1 423173-100G
mol (D.S.)
Propylene oxide 0.1–0.3 mol
per 1 mol (M.S.)
Methoxy 21 wt. %
Propylene oxide 5 wt. %
Methyl 2-hydroxyethyl RO - Methyl 1.3–2.2 mol per 1 435015-250G
cellulose mol (D.S.)
OO R Hydroxyethyl 0.06–0.50 mol
RO OR per 1 mol (M.S.)
R = H or CH3 or
OO Methoxy 26 wt. %
OR OR * H Hydroxyethyl 8 wt. %
O
x
RO OR n

Sodium carboxymethyl RO Average Mw ~250,000 Carboxymethyl groups 0.7 419311-100G

cellulose 419311-1KG
OO R
RO OR Average Mw ~250,000 Carboxymethyl groups 0.9 419303-100G
R = H or
419303-1KG
OO O
OR OR Average Mw ~250,000 Carboxymethyl groups 1.2 419281-100G
* 419281-1KG
ONa
RO OR n Average Mw ~700,000 Carboxymethyl groups 0.9 419338-100G
419338-1KG
 3D Bioprinting: Printing a Bright Future
SigmaAldrich.com/matsci 57

Chitosans
Name Molecular Weight Description Cat. No.
Chitosan 50–190 kDa Low molecular weight 448869-50G
448869-250G
190–375 kDa - 417963-25G
417963-100G
- Medium molecular weight 448877-50G
448877-250G
310–375 kDa High molecular weight 419419-50G
419419-250G
Average Mw 50 kDa Biological source: Fungal fermentation 900341-2G
High purity
Non-animal derived
Average Mw 100 kDa High purity 900342-2G
Non-animal derived
Chitosan-mPEG 1k Medium MW 40–70% PEGylation 923834-1G
Chitosan oligosaccharide lactate Average Mn 5,000 - 523682-1G
523682-10G
Trimethyl chitosan Low molecular weight Degree of quaternization >70% 912700-1G
Medium molecular weight Degree of quaternization: 40–60% 912123-1G
High molecular weight Degree of quaternization >70% 912034-1G

Lignins
Name Molecular Weight Solubility Cat. No.
Lignin, alkali Average Mw ~10,000 - 471003-100G
471003-500G
- Ethylene glycol soluble 370959-100G
NaOH 0.05 % (warm 5% aquesous) 370959-500G
Benzene insoluble
Methanol partially soluble
Dioxane soluble
Hexane insoluble
MEK partially soluble
Lignosulfonic acid calcium salt Average Mn ~2,500 H2O soluble 471054-100G
Average Mw ~18,000
Lignosulfonic acid sodium salt Average Mn ~7,000 H2O soluble 471038-100G
Average Mw ~52,000 471038-500G

Hyaluronic Acid
Name MW Description Cat. No.
Hyaluronic acid methacrylate 20,000–30,000 NMR: Conforms to structure 914568-500MG
degree of substitution 20–50%
50,000–70,000 NMR: Conforms to structure 914304-500MG
degree of substitution 20–50%
120,000–150,000 NMR: Conforms to structure 914800-500MG
degree of substitution: 20–50%
58 Bioinks

Bioinks
TissueFab®
Name Description Composition Low Endo Bioburden Cat. No.
TissueFab® bioink Crosslinking solution, low CaCl2 Yes Yes 919926-1EA
endotoxin
(Gel)ma -UV/365 nm GelMA - Yes 905429-1EA
(Gel)ma -Vis/405 nm, low GelMA Yes Yes 918741-1EA
endotoxin
Alg(Gel)ma -UV/365 nm GelMA, Alginate - Yes 905410-10ML
Alg(Gel)ma -Vis/525 nm GelMA, Alginate - Yes 906913-1EA
(GelAlg)ma -UV/365 nm GelMA, AlgMA - Yes 920983-1EA
(GelAlg)ma -Vis/405 nm GelMA, AlgMA - Yes 921610-1EA
(GelHA)ma -UV/365 nm GelMA, HAMA - Yes 919632-1EA
(GelHA)ma -Vis/405 nm GelMA, HAMA - Yes 919624-1EA
(GelAlgHA)ma -UV/365 nm GelMA, AlgMA, HAMA - Yes 920975-1EA
(GelAlgHA)ma -Vis/405 nm GelMA, AlgMA, HAMA - Yes 922862-1EA
Sacrificial Pluronic - Yes 906905-1EA
TissueFab® bioink Bone UV/365 nm GelMA, Hydroxyapetite - Yes 915025-1EA
Vis/405 nm GelMA, Hydroxyapetite - Yes 915033-1EA
Support gel PCL, Hydroxyapetite - - 915637-5G
TissueFab® bioink Conductive UV/365 nm GelMA, CNTs - Yes 915726-1EA
Vis/405 nm GelMA, CNTs - Yes 915963-1EA

Gelatin Methacryloyl (GelMA)

Name Gel Strength (g Bloom) Degree Of Functionalization Cat. No.
Gelatin methacryloyl 90-110 Degree of substitution: 60% 900628-1G
170–195 Degree of substitution: 60% 900741-1G
300 Degree of substitution: 40% 900629-1G
900629-5G
300 Degree of substitution: 60% 900622-1G
300 Degree of substitution: 80% 900496-1G

Modified Gelatins
Name Gel Strength (g Bloom) Degree Of Functionalization Cat. No.
Allyl-modified gelatin 300 Degree of substitution: 70% by TNBS method 901553-1G
Azide functionalized gelatin - Degree of substitution: greater than 80% by TNBS method 907723-1G
NMR: Conforms to structure
Degree of substitution >80%
Gelatin-Rhodamine B 300 1–10 μmol Rhodamine B per g gelatin 923869-1G
mPEG functionalized gelatin 300 50% PEGylation 920444-1G
Thiol functionalized gelatin - NMR: Conforms to structure 904643-1G
Thiol content 200–300 μmol/g

Low Endotoxins
Name Description Form Impurities Cat. No.
Low endotoxin alginate Medium viscosity Lyophilized powder Bioburden <10 CFU/g 919373-1EA
Endotoxin <100 EU/g
Low endotoxin alginate Medium viscosity Viscous liquid Bioburden <5 CFU/g Total Aerobic 918652-1EA
solution Bioburden <5 CFU/g Fungal
Endotoxin <10 EU/g
Low endotoxin gelatin from - Powder Endotoxin ≤10 EU/g 920037-1G
bovine bone
Low endotoxin gelatin from - Powder Endotoxin <10 EU/g 901757-1G
porcine skin Total viable aerobic count <300 g 901757-5G
total impurities <10 EU/g
- Powder Endotoxin <10 EU/g 901756-1G
Total viable aerobic count <300 g 901756-5G
- Powder Endotoxin ≤10 EU/g 920010-1G
Low endotoxin gelatin - Viscous liquid Bioburden <5 cfu/mL 918644-1EA
solution Endotoxin <25 EU/mL
Low endotoxin GelMA Degree of substitution 80% Powder, chunks, or fibers Bioburden <10 CFU/g 918628-1EA
Endotoxin <125 EU/g
Degree of substitution 60% Powder Endotoxin ≤10 EU/g 920045-1G
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Name Description Form Impurities Cat. No.

Low endotoxin GelMA Degree of substitution 80% Viscous liquid Bioburden <5 cfu/mL 918636-1EA
solution Endotoxin <25 EU/mL
Low endotoxin non-gelling - Powder Endotoxin ≤10 EU/g 920029-1G
gelatin from porcine skin

Alginate-Based Bioinks
Name Form Impurities pH Cat. No.
Alginate bioink Viscous liquid Endotoxin <25 EU/mL 6.5–7 901953-1EA
Alginate-RGD bioink Viscous liquid Endotoxin <25 EU/mL 6.5–7 901950-1EA
Cellulose-Alginate bioink Viscous liquid Endotoxin <25 EU/mL 6.5–7 901960-1EA
Cellulose-Alginate-Calcium Phosphate bioink Viscous liquid Endotoxin <25 EU/mL 6.5–7 901958-1EA
Cellulose-Alginate-RGD bioink Viscous liquid Endotoxin <25 EU/mL 6.5–7 901955-1EA

Decellularized Bioinks
Name Description (μg/mg) Form Cat. No.
Decellularized ECM bioink precursor from porcine skin GAG (biocolor) 0.4-0.8 Semisolid 906867-1EA
Collagen (hydroxyproline) 90-125
Decellularized ECM bioink precursor from porcine bone GAG (bicolor) 1.5-5.0 Semisolid 906883-1EA
Collagen (hydroxyproline) 60-120
Decellularized ECM bioink precursor from porcine cartilage GAG (biocolor) 2.0-6.0 Semisolid 906875-1EA
Collagen (hydroxyproline) 60-120

Collagen Bioinks
Name Description Kit Components Endotoxin (EU/mL) Cat. No.
Lifeink® 200 Neutralized type I collagen bioink Sterile-filtered yes ≤10 916226-1EA
Lifeink® 240 Acidic type I collagen bioink Sterile-filtered yes ≤10 915211-1EA
LifeSupport™ Support slurry for FRESH bioprinting Irradiated yes - 915467-1EA
PhotoCol™-IRG, Methacrylated collagen: Sterile-filtered yes ≤10 917575-1EA
methacrylated collagen Degree of methacrylation ≥ 20% Methacrylated collagen (100 mg)
bioink kit, with Irgacure 20 mM acetic acid (50 mL)
Neutralization solution (10 mL)
Irgacure photoinitiator ( 100 mg)
PhotoCol™-LAP Methacrylated collagen: Sterile-filtered yes ≤10 916293-1EA
Degree of methacrylation ≥ 20% Methacrylated collagen (100 mg)
20 mM acetic acid (50 mL)
Neutralization solution (10 mL)
LAP photoinitiator (100 mg)
Methacrylated collagen bioink kit, with LAP
PhotoCol™-RUT, Methacrylated collagen: Sterile-filtered yes ≤10 917834-1EA
methacrylated collagen Degree of methacrylation ≥ 20% Methacrylated collagen (100 mg)
bioink kit, with 20 mM acetic acid (50 mL)
ruthenium Neutralization solution (10 mL)
Ruthenium (100 mg)
Sodium persulfate photoinitiator (500 mg)

HA Bioinks
Name MW (kDa) Degree of Methacrylation Kit Components Cat. No.
PhotoHA™-IRG, methacrylated 100–150 ≥ 45-65% Methacrylated hyaluronic acid (100 mg) 917079-1EA
hyaluronic acid bioink kit, with Irgacure Irgacure photoinitiator (100 mg)
PhotoHA™-LAP, methacrylated 100–150 ≥45-65% Methacrylated hyaluronic acid (100 mg) 916471-1EA
hyaluronic acid bioink kit, with LAP LAP photoinitiator (100 mg)
PhotoHA™-RUT 100–150 ≥ 45-65% Methacrylated hyaluronic acid (100 mg) 917338-1EA
Ruthenium (100 mg)
Sodium persulfate photoinitiator (500 mg)
Methacrylated hyaluronic acid bioink kit, with
ruthenium

Ready-Made Bioinks
Name pH Viscosity Form Impurities (LB Broth) Cat. No.
INVIVO-GEL BMP2 bioink 8–9 >30K cP Opaque gel ND CFU/mL 923745-1EA
INVIVO-GEL essential bioink 8–9 >30K cP Opaque gel ND CFU/mL 923796-1EA
INVIVO-GEL TGF-β bioink 8–9 >30K cP Opaque gel ND CFU/mL 923753-1EA
INVIVO-GEL VEGF bioink 8–9 >30K cP Opaque gel ND CFU/mL 923737-1EA
60 Bioinks

Biodegradable Polymers
Name Structure Molecular Weight Cat. No.
Bio-based Polyether H Mw 400‑600 Da 923990-500G
HO O
Polyol n 923990-1KG
Mw 900‑1100 Da 923974-1KG
923974-500G
Mw 1900‑2100 Da 923966-500G
923966-1KG
2600‑2800 Da 923982-500G
923982-1KG
Polycaprolactone O Average Mn ~10,000 by GPC 440752-5G
O Average Mw ~14,000 440752-250G
n
440752-500G

Polycaprolactone O O Average Mn 5,000 914495-1G

diacrylate O O O Average Mn 10,000 914509-1G
O O n
n
O O

Polycaprolactone O O Average Mn 3,000 802158-2G

dimethacrylate O O O Average Mn 5,000 914762-1G
O O n
n Average Mn 10,000 915106-1G
O O

Polylactic acid O Mn ~30,000 38534-1G

Mw ~60,000 38534-5G
O
CH3 n

Poly(l-lactide), acrylate O Average Mn 5,500 775983-1G

terminated
O O
H O CH2
CH3 n
O

Poly(d,l-lactide-co- O - 457647-5G
caprolactone) O
O
CH3 O
x y

Poly(l-lactide) O O Average Mn 10,000 916102-1G

dimethacrylate O O O
O O
n n
O O

O O Average Mn 5,000 915009-1G

O O O
O O n
n
O O

Resomer® R 202 H, O Mw 10,000‑18,000 719978-1G

Poly(d,l-lactide) 719978-5G
O
CH3
n

Resomer® RG 502 H, O Mw 7,000–17,000 719897-1G

Poly(d,l-lactide-co- O 719897-5G
glycolide) O
CH3 O
x y

Poly(ethylene glycol) and Poly(ethylene oxide)

Name Structure Avg. Mn (Da) Cat. No.
4-Arm-PEG20K-acrylate O Average Mn 20,000 JKA7034-1G
H2C O O
O O O CH2
n n O
O
O O
O O
H2C O O O O CH2
n
n

8-Arm-PEG10K-acrylate, O Average Mn 10,000 JKA10021-1G

tripentaerythritol core
R O CH2
O n O
8

R = tripentearythritol core structure

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Name Structure Avg. Mn (Da) Cat. No.

4-Arm PEG10K-Azide - JKA7163-1G
R O N3
O n 4
R = Pentaerythritol core structure

Poly(ethylene glycol) O O PEG average Mn10,000 901635-500MG

bis(2-pyridyl KAT) PEG ~10,000 Da
KF3B BF3K
N O N
O n O

Poly(ethylene glycol)- O CH2 Average Mn ~5,800 915858-5G

block-poly(propylene O O 915858-1G
glycol)-block- O O
CH2 y z O
Average Mn ~8,400 914428-5G
poly(ethylene glycol) x
914428-1G
diacrylate
Average Mn ~14,600 914665-5G
914665-1G
Poly(ethylene glycol)- O Average Mn ~12,500 914169-1G
block-poly(propylene O O 914169-5G
glycol)-block- O O
Average Mn ~14,600 913901-5G
poly(ethylene glycol) y z O 913901-1G
dimethacrylate
x

Poly(ethylene glycol) O PEG average Mn20,000 (n~450) 767549-1G

diacrylate O CH2 Average Mn 20,000
H2C O
Poly(ethylene glycol) O n Average Mn 6,000 687537-1G
dimethacrylate

Bioprinting Consumables
Consumables
Name Description Cat. No.
Empty cartridges 3mL cartridges (50/box), amber, for UV and light-sensitive materials (up to 550 nm) 917257-1EA
3mL cartridges (50/box), clear 917753-1EA
3mL cartridges (50/box), opaque black, for complete light blockage 917516-1EA
End caps for cartridges End caps (50/box), blue,suitable for 3mL cartridge 917001-1EA
Pistons 3mL pistons (50/box), white, suitable for 3mL cartridge 916749-1EA
Stainless steel dispensing tips Stainless steel dispensing tips, 20 gauge, 0.50", pink, 50/box 917028-1EA
Stainless steel dispensing tips, 21 Gauge, 0.50", purple, 50/box 916757-1EA
Stainless steel dispensing tips, 22 gauge, 0.50", blue, 50/box 918024-1EA
Stainless steel dispensing tips, 23 gauge, 0.50", orange, 50/box 917761-1EA
Stainless steel dispensing tips, 25 gauge, 0.50", red, 50/box 917036-1EA
Stainless steel dispensing tips, 27 gauge, 0.50", clear, 50/box 917532-1EA
Tapered dispensing tips Tapered dispensing tips, polypropylene, 20 gauge, pink, ID 0.023", 50/box 917273-1EA
Tapered dispensing tips, polypropylene, 22 gauge, blue, ID 0.016", 50/box 917265-1EA
Tapered dispensing tips, polypropylene, 25 gauge, red, ID 0.010", 50/box 916765-1EA
Tapered dispensing tips, polypropylene, 27 gauge, clear, ID 0.008", 50/box 917524-1EA
Tip caps for cartridges Suitable for 3mL cartridge one size, blue, 50/box 916773-1EA
Air adaptor Air adaptor, for 3 mL Cartridge, 1 EA 918016-1EA

Notes:
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Frankfurter Strasse 250
64293 Darmstadt, Germany

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